Packaging of Immunostimulatory Substances Into Virus-Like Particles: Method of Preparation and Use

ABSTRACT

The invention relates to the finding that virus like particles (VLPs) can be loaded with immunostimulatory substances, in particular with DNA oligonucleotides containing non-methylated C and G (CpGs). Such CpG-VLPs are dramatically more immunogenic than their CpG-free counterparts and induce enhanced B and T cell responses. The immune response against antigens optionally coupled, fused or attached otherwise to the VLPs is similarly enhanced as the immune response against the VLP itself. In addition, the T cell responses against both the VLPs and antigens are especially directed to the Th1 type. Antigens attached to CpG-loaded VLPs may therefore be ideal vaccines for prophylactic or therapeutic vaccination against allergies, tumors and other self-molecules and chronic viral diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/457,348, filed Mar. 26, 2003, which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to the fields of vaccinology,immunology and medicine. The invention provides compositions and methodsfor enhancing immunological responses against virus-like particles(VLPs) or against antigens coupled, fused or attached otherwise to VLPsby packaging immunostimulatory substances, in particularimmunostimulatory nucleic acids, and even more particularoligonucleotides containing at least one non-methylated CpG sequence,into the VLPs. The invention can be used to induce strong and sustainedT cell responses particularly useful for the treatment of tumors andchronic viral diseases as well as allergies and other chronic diseases.

2. Related Art

The essence of the immune system is built on two separate foundationpillars: one is specific or adaptive immunity which is characterized byrelatively slow response-kinetics and the ability to remember; the otheris non-specific or innate immunity exhibiting rapid response-kineticsbut lacking memory.

It is well established that the administration of purified proteinsalone is usually not sufficient to elicit a strong immune response;isolated antigen generally must be given together with helper substancescalled adjuvants. Within these adjuvants, the administered antigen isprotected against rapid degradation, and the adjuvant provides anextended release of a low level of antigen.

Unlike isolated proteins, viruses induce prompt and efficient immuneresponses in the absence of any adjuvants both with and without T-cellhelp (Bachmann & Zinkernagel, Ann. Rev. Immunol. 15:235-270 (1997)).Many viruses exhibit a quasi-crystalline surface that displays a regulararray of epitopes which efficiently crosslinks epitope-specificimmunoglobulins on B cells (Bachmann & Zinkernagel, Immunol. Today17:553-558 (1996)). Viral structure is even linked to the generation ofanti-antibodies in autoimmune disease and as a part of the naturalresponse to pathogens (see Fehr, T., et al., J. Exp. Med. 185:1785-1792(1997)). Thus, antigens on viral particles that are organized in anordered and repetitive array are highly immunogenic since they candirectly activate B cells and induce the generation of a cytotoxic Tcell response, another crucial arm of the immune system.

Viral particles as antigens exhibit two advantages over their isolatedcomponents: (1) due to their highly repetitive surface structure, theyare able to directly activate B cells, leading to high antibody titersand long-lasting B cell memory; and (2) viral particles, but not solubleproteins, have the potential to induce a cytotoxic T cell response, evenif the viruses are non-infectious and adjuvants are absent.

Several new vaccine strategies exploit the inherent immunogenicity ofviruses. Some of these approaches focus on the particulate nature of thevirus particle; for example see Harding, C. V. and Song, R., (J.Immunology 153:4925 (1994)), which discloses a vaccine consisting oflatex beads and antigen; Kovacsovics-Bankowski, M., et al. (Proc. Natl.Acad. Sci. USA 90:4942-4946 (1993)), which discloses a vaccineconsisting of iron oxide beads and antigen; U.S. Pat. No. 5,334,394 toKossovsky, N., et al., which discloses core particles coated withantigen; U.S. Pat. No. 5,871,747, which discloses synthetic polymerparticles carrying on the surface one or more proteins covalently bondedthereto; and a core particle with a non-covalently bound coating, whichat least partially covers the surface of said core particle, and atleast one biologically active agent in contact with said coated coreparticle (see, e.g., WO 94/15585).

In a further development, virus-like particles (VLPs) are beingexploited in the area of vaccine production because of both theirstructural properties and their non-infectious nature. VLPs aresupermolecular structures built in a symmetric manner from many proteinmolecules of one or more types. They lack the viral genome and,therefore, are noninfectious. VLPs can often be produced in largequantities by heterologous expression and can be easily be purified.

In addition, DNA rich in non-methylated CG motifs (CpG), as present inbacteria and most non-vertebrates, exhibits a potent stimulatoryactivity on B cells, dendritic cells and other APC's In vitro as well asin vivo. Although bacterial DNA is immunostimulatory across manyvertebrate species, the individual CpG motifs may differ. In fact, CpGmotifs that stimulate mouse immune cells may not necessarily stimulatehuman immune cells and vice versa.

Although DNA oligomers rich in CpG motifs can exhibit immunostimulatorycapacity, their efficiency is often limited, since they are unstable invitro and in vivo. Thus, they exhibit unfavorable pharmacokinetics. Inorder to render CpG-oligonucleotides more potent, it is thereforeusually necessary to stabilize them by introducing phosphorothioatemodifications of the phosphate backbone.

A second limitation for the use of CpG-oligonucleotides to stimulateimmune responses is their lack of specificity, since all APC's and Bcells in contact with CpG-oligonucleotides become stimulated. Thus, theefficiency and specificity of CpG-oligonucleotides may be improved bystabilizing them or packaging them in a way that restricts cellularactivation to those cells that also present the relevant antigen.

In addition, immunostimulatory CpG-oligodeoxynucleotides induce strongside effects by causing extramedullary hemopoiesis accomponied bysplenomegaly and lymphadenopathy in mice (Sparwasser et al., J. Immunol.(1999), 162:2368-74 and Example 18).

VLPs have been shown to be efficiently presented on MHC class Imolecules as they, presumably after uptake by macropinocytosis, areefficiently processed and crossprimed onto MHC class I. The mechanism ofcrosspriming is not clear to date, but TAP-dependent and TAP-independentpathways have been proposed.

There have been remarkable advances made in vaccination strategiesrecently, yet there remains a need for improvement on existingstrategies. In particular, there remains a need in the art for thedevelopment of new and improved vaccines that promote a strong CTLimmune response and anti-pathogenic protection as efficiently as naturalpathogens in the absence of generalized activation of APCs and othercells.

SUMMARY OF THE INVENTION

This invention is based on the surprising finding that specificimmunostimulatory substances such as DNA oligonucleotides packaged intoVLPs renders them more immunogenic. Unexpectedly, the nucleic acids andoligonucleotides, respectively, present in VLPs can be replacedspecifically by the immunostimulatory substances andDNA-oligonucleotides containing CpG motifs, respectively. Surprisingly,these packaged immunostimulatory substances, in particularimmunostimulatory nucleic acids such as unmethylated CpG-containingoligonucleotides retained their immunostimulatory capacity withoutwidespread activation of the innate immune system. The compositionscomprising VLP's and the immunostimulatory substances in accordance withthe present invention, and in particular the CpG-VLPs are dramaticallymore immunogenic than their CpG-free counterparts and induce enhanced Band T cell responses. The immune response against antigens optionallycoupled, fused or attached otherwise to the VLPs is similarly enhancedas the immune response against the VLP itself. In addition, the T cellresponses against both the VLPs and antigens are especially directed tothe Th1 type. Antigens attached to CpG-loaded VLPs may therefore beideal vaccines for prophylactic or therapeutic vaccination againstallergies, tumors and other self-molecules and chronic viral diseases.

In a first embodiment, the invention provides a composition, typicallyand preferably for enhancing an immune response in an animal, comprisinga virus-like particle and an immunostimulatory substance, preferably animmunostimulatory nucleic acid, an even more preferably an unmethylatedCpG-containing oligonucleotide, where the substance, nucleic acid oroligonucleotide is coupled, fused, or otherwise attached to or enclosedby, i.e., bound, to the virus-like particle. In another embodiment, thecomposition further comprises an antigen bound to the virus-likeparticle.

In a preferred embodiment of the invention, the immunostimulatorynucleic acids, in particular the unmethylated CpG-containingoligonucleotides are stabilized by phosphorothioate modifications of thephosphate backbone. In another preferred embodiment, theimmunostimulatory nucleic acids, in particular the unmethylatedCpG-containing oligonucleotides are packaged into the VLPs by digestionof RNA within the VLPs and simultaneous addition of the DNAoligonucleotides containing CpGs of choice. In an equally preferredembodiment, the VLPs can be disassembled before they are reassembled inthe presence of CpGs.

In a further preferred embodiment, the immunostimulatory nucleic acidsdo not contain CpG motifs but nevertheless exhibit immunostimulatoryactivities. Such nucleic acids are described in WO 01/22972. Allsequences described therein are hereby incorporated by way of reference.

In a further preferred embodiment, the virus-like particle is arecombinant virus-like particle. Also preferred, the virus-like particleis free of a lipoprotein envelope. Preferably, the recombinantvirus-like particle comprises, or alternatively consists of, recombinantproteins of Hepatitis B virus, BK virus or other human Polyoma virus,measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth-Disease virus,Retrovirus, Norwalk virus or human Papilloma virus, RNA-phages,Qβ-phage, GA-phage, fr-phage and Ty. In a specific embodiment, thevirus-like particle comprises, or alternatively consists of, one or moredifferent Hepatitis B virus core (capsid) proteins (HBcAgs).

In a further preferred embodiment, the virus-like particle comprisesrecombinant proteins, or fragments thereof, of a RNA-phage. PreferredRNA-phages are Qβ-phage, AP 205-phage, GA-phage, fr-phage

In another embodiment, the antigen is a recombinant antigen. In yetanother embodiment, the antigen can be selected from the groupconsisting of: (1) a polypeptide suited to induce an immune responseagainst cancer cells; (2) a polypeptide suited to induce an immuneresponse against infectious diseases; (3) a polypeptide suited to inducean immune response against allergens; (4) a polypeptide suited to inducean improved response against self-antigens; and (5) a polypeptide suitedto induce an immune response in farm animals or pets.

In yet another embodiment, the antigen can be selected from the groupconsisting of: (1) an organic molecule suited to induce an immuneresponse against cancer cells; (2) an organic molecule suited to inducean immune response against infectious diseases; (3) an organic moleculesuited to induce an immune response against allergens; (4) an organicmolecule suited to induce an improved response against self-antigens;(5) an organic molecule suited to induce an immune response in farmanimals or pets; and (6) an organic molecule suited to induce a responseagainst a drug, a hormone or a toxic compound.

In a particular embodiment, the antigen comprises, or alternativelyconsists of, a cytotoxic T cell epitope, preferably a Th cell epitope ora combination of at least two of the epitopes, wherein preferably the atleast two epitopes are bound directly or by way of a linking sequence.In one embodiment, the cytotoxic T cell epitope is a viral or a tumorcytotoxic T cell epitope. In a related embodiment, the virus-likeparticle comprises the Hepatitis B virus core protein and the cytotoxicT cell epitope is fused to the C-terminus of said Hepatitis B virus coreprotein, preferably by way of a linking sequence. In another embodiment,the virus-like particle comprises the BK virus VP1 protein and thecytotoxic T cell epitope is fused to the C-terminus of the BK virus VP1protein, preferably by way of a linking sequence. In one embodiment,they are fused by a leucine linking sequence.

In another aspect of the invention, there is provided a method ofenhancing an immune response in a human or other animal speciescomprising introducing into the animal a composition comprising avirus-like particle and immunostimulatory substance, preferably animmunostimulatory nucleic acid, an even more preferably an unmethylatedCpG-containing oligonucleotide where the substance, preferably thenucleic acid, and even more preferably the oligonucleotide is bound(i.e. coupled, attached or enclosed) to the virus-like particle. In afurther embodiment, the composition further comprises an antigen boundto the virus-like particle.

In yet another embodiment of the invention, the composition isintroduced into an animal subcutaneously, intramuscularly, intranasally,intradermally, intravenously or directly into a lymph node. In anequally preferred embodiment, the immune enhancing composition isapplied locally, near a tumor or local viral reservoir against which onewould like to vaccinate.

In a preferred aspect of the invention, the immune response is a T cellresponse, and the T cell response against the antigen is enhanced. In aspecific embodiment, the T cell response is a cytotoxic T cell response,and the cytotoxic T cell response against the antigen is enhanced.

The present invention also relates to a vaccine comprising animmunologically effective amount of the immune enhancing composition ofthe present invention together with a pharmaceutically acceptablediluent, carrier or excipient. In a preferred embodiment, the vaccinefurther comprises at least one adjuvant, such as incomplete Freund'sadjuvant. The invention also provides a method of immunizing and/ortreating an animal comprising administering to the animal animmunologically effective amount of the disclosed vaccine.

In a preferred embodiment of the invention, the immunostimulatorysubstance-containing VLPs, preferably the immunostimulatory nucleicacid-containing VLP's, an even more preferably the unmethylatedCpG-containing oligonucleotide VLPs are used for vaccination of animalsor humans against the VLP itself or against antigens coupled, fused orattached otherwise to the VLP. The modified VLPs can be used tovaccinate against tumors, viral diseases, self-molecules and selfantigens, respectively, or non-peptidic small molecules, for example.The vaccination can be for prophylactic or therapeutic purposes, orboth. Also, the modified VLPs can be used to vaccinate against allergiesin order to induce immune-deviation.

In the majority of cases, the desired immune response will be directedagainst antigens coupled, fused or attached otherwise to theimmunostimulatory substance-containing VLPs, preferably theimmunostimulatory nucleic acid-containing VLP's, an even more preferablythe unmethylated CpG-containing oligonucleotide VLPs. The antigens canbe peptides, proteins, domains, carbohydrates or small molecules suchas, for example, steroid hormones or drugs, such as nicotine. Under someconditions, the desired immune response can be directed against the VLPitself. This latter application will be used in cases where the VLPoriginates from a virus against which one would like to vaccinate.

The route of injection is preferably subcutaneous or intramuscular, butit would also be possible to apply the CpG-containing VLPsintradermally, intranasally, intravenously or directly into the lymphnode. In an equally preferred embodiment, the CpG-containingantigen-coupled or free VLPs are applied locally, near a tumor or localviral reservoir against which one would like to vaccinate.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide further explanation of the invention asclaimed.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows the SDS-PAGE analysis of Qb-MelanA VLPs. MelanA-peptideswere coupled to Qb VLPs, as described in Example 22. The final productswere mixed with sample buffer and separated under reduced conditions on16% Novex®Tris-Glycine gels for 1.5 hours at 125 V. The separatedproteins were stained by soaking the gel in Coomassie blue solution.Background staining was removed by washing the gel in 50% methanol, 8%acetic acid. The Molecular weight marker (P 77085, New England BioLabs,Beverly, USA) was used as reference for Qb-MelanA migration velocity(lane 1). 14 μg of either Qb alone (lane 2) or Qb derivatized with SMPH(lane 3) were loaded for comparison with 8 μg of each final product:Qb-MelanA 16-35 (lane 4), Qb-MelanA 16-35 A/L (lane 5), Qb-MelanA 26-35(lane 6) and Qb-MelanA 26-35 A/L (lane 7).

FIG. 2A shows IFN alpha released in the supernatants of ISS-treatedhuman PBMC. PBMC were obtained from buffy coat and incubated withfivefold dilution of the indicated ISS for 18 h. The term G10 is usedfor the the oligonucleotide G10-PO, and the term G3 is used for theoligonucleotide G3-6). Supernatants were collected and IFN alpha wasmeasured by ELISA, using a set of antibodies provided by PBL BiomedicalLaboratories.

FIG. 2B shows the upregulation of CD69 on human CD8+ PBMC treated withISS. PBMC were obtained from buffy coat and incubated with fivefolddilution of the indicated ISS for 18 h. Cells were washed and incubatedwith anti-CD8-FITC, anti-CD19-PE and anti-CD69-APC (all from BDPharMingen) for 20 min on ice. After washing, cells were analysed on aFACS Calibur using CellQuest software.

FIG. 3 shows the virus titers after immunizing mice with Qbx33 packagedwith poly (I:C), G3-6, or G6. C57B16 mice were immunized by injectingeither 100 μg Qbx33, 100 μg Qb VLPs packaged with poly (I:C) and coupledto p33 (Qb-pIC-33, also termed QbxZnxpolyICxp33GGC), 90 μg Qbx33packaged with G3-6 (Qbx33/G3-6), or 90 μg Qbx33 packaged with G6(Qbx33/G6). After eight days, mice were challenged with 1.5×106 plaqueforming units Vaccinia virus, carrying the LCMV-p33 epitope. Five dayslater, mice were sacrificed and the ovaries were collected. A singlecell suspension from the ovaries was prepared and added to BCS40 cellsin serial dilutions. One day later, the cell layer was stained with asolution containing 50% Ethanol, 2% formaldehyde, 0.8% NaCl and 0.5%Crystal violet) and the viral plaques were counted.

FIG. 4 shows the SDS-PAGE analysis of the coupling reaction of Qβ VLP togag-G50 peptide. The samples were run under reducing conditions on a 12%NuPage gel (Invitrogen). Lane 1 is the protein marker, withcorresponding molecular weights indicated on the left border of the gel;lane 2, derivatized Qβ VLP; lane 3, the supernatant of the couplingreaction of Qβ capsid protein to the gag-G50 peptide; lane 4, the pelletof the coupling reaction of Qβ capsid protein to the gag-G50 peptide.Coupling products corresponding to the coupling of a peptide on a Qβmonomer or Qβ dimer are indicated by arrows in the Figure.

FIG. 5 shows the SDS-PAGE analysis of the coupling reaction of Qβ VLP tonef-N56 peptide. The samples were run under reducing conditions on a 12%NuPage gel (Invitrogen). Lane 1 is the protein marker, withcorresponding molecular weights indicated on the left border of the gel;lane 2, derivatized Qβ VLP; lane 3, the supernatant of the couplingreaction of Qβ capsid protein to the nef-N56 peptide; lane 4, the pelletof the coupling reaction of Qβ capsid protein to the nef-N56 peptide.Coupling products corresponding to the coupling of a peptide on a Qβmonomer or Qβ dimer are indicated by arrows in the Figure.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are hereinafter described.

1. Definitions

Amino acid linker: An “amino acid linker”, or also just termed “linker”within this specification, as used herein, either associates the antigenor antigenic determinant with the second attachment site, or morepreferably, already comprises or contains the second attachment site,typically—but not necessarily—as one amino acid residue, preferably as acysteine residue. The term “amino acid linker” as used herein, however,does not intend to imply that such an amino acid linker consistsexclusively of amino acid residues, even if an amino acid linkerconsisting of amino acid residues is a preferred embodiment of thepresent invention. The amino acid residues of the amino acid linker are,preferably, composed of naturally occurring amino acids or unnaturalamino acids known in the art, all-L or all-D or mixtures thereof.However, an amino acid linker comprising a molecule with a sulfhydrylgroup or cysteine residue is also encompassed within the invention. Sucha molecule comprise preferably a C1-C6 alkyl-, cycloalkyl (C5,C6), arylor heteroaryl moiety. However, in addition to an amino acid linker, alinker comprising preferably a C1-C6 alkyl-, cycloalkyl- (C5,C6), aryl-or heteroaryl- moiety and devoid of any amino acid(s) shall also beencompassed within the scope of the invention, Association between theantigen or antigenic determinant or optionally the second attachmentsite and the amino acid linker is preferably by way of at least onecovalent bond, more preferably by way of at least one peptide bond.

Animal: As used herein, the term “animal” is meant to include, forexample, humans, sheep, horses, cattle, pigs, dogs, cats, rats, mice,mammals, birds, reptiles, fish, insects and arachnids.

Antibody: As used herein, the term “antibody” refers to molecules whichare capable of binding an epitope or antigenic determinant. The term ismeant to include whole antibodies and antigen-binding fragments thereof,including single-chain antibodies. Most preferably the antibodies arehuman antigen binding antibody fragments and include, but are notlimited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv),single-chain antibodies, disulfide-linked Fvs (sdFv) and fragmentscomprising either a VL or VH domain. The antibodies can be from anyanimal origin including birds and mammals. Preferably, the antibodiesare human, murine, rabbit, goat, guinea pig, camel, horse or chicken. Asused herein, “human” antibodies include antibodies having the amino acidsequence of a human immunoglobulin and include antibodies isolated fromhuman immunoglobulin libraries or from animals transgenic for one ormore human immunoglobulins and that do not express endogenousimmunoglobulins, as described, for example, in U.S. Pat. No. 5,939,598by Kucherlapati et al.

Antigen: As used herein, the term “antigen” refers to a molecule capableof being bound by an antibody or a T cell receptor (TCR) if presented byMHC molecules. The term “antigen”, as used herein, also encompassesT-cell epitopes. An antigen is additionally capable of being recognizedby the immune system and/or being capable of inducing a humoral immuneresponse and/or cellular immune response leading to the activation of B-and/or T-lymphocytes. This may, however, require that, at least incertain cases, the antigen contains or is linked to a T helper cellepitope (Th cell epitope) and is given in adjuvant. An antigen can haveone or more epitopes (B- and T-epitopes). The specific reaction referredto above is meant to indicate that the antigen will preferably react,typically in a highly selective manner, with its corresponding antibodyor TCR and not with the multitude of other antibodies or TCRs which maybe evoked by other antigens. Antigens as used herein may also bemixtures of several individual antigens.

A “microbial antigen” as used herein is an antigen of a microorganismand includes, but is not limited to, infectious virus, infectiousbacteria, parasites and infectious fungi. Such antigens include theintact microorganism as well as natural isolates and fragments orderivatives thereof and also synthetic or recombinant compounds whichare identical to or similar to natural microorganism antigens and inducean immune response specific for that microorganism. A compound issimilar to a natural microorganism antigen if it induces an immuneresponse (humoral and/or cellular) to a natural microorganism antigen.Such antigens are used routinely in the art and are well known to theskilled artisan.

Examples of infectious viruses that have been found in humans includebut are not limited to: Retroviridae (e.g. human immunodeficiencyviruses, such as HIV-1 (also referred to as HTLV-III, LAV orHTLV-III/LAV, or HIV-III); and other isolates, such as HIV-LP);Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses,human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.strains that cause gastroenteritis); Togaviridae (e.g. equineencephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses,encephalitis viruses, yellow fever viruses); Coronoviridae (e.g.coronaviruses); Rhabdoviradae (e.g. vesicular stomatitis viruses, rabiesviruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g.parainfluenza viruses, mumps virus, measles virus, respiratory syncytialvirus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g.Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis Bvirus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses,polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,cytomegalovirus (CMV), herpes virus); Poxviridae (variola viruses,vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swinefever virus); and unclassified viruses (e.g. the etiological agents ofSpongiform encephalopathies, the agent of delta hepatitis (thought to bea defective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e. Hepatitis C); Norwalk and related viruses, andastroviruses).

Both gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to, Pasteurella species, Staphylococci species and Streptococcusspecies. Gram negative bacteria include, but are not limited to,Escherichia coli, Pseudomonas species, and Salmonella species. Specificexamples of infectious bacteria include but are not limited to:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps. (e.g. M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfluenzae, Bacillus antracis, Corynebacterium diphtheriae,Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Rickettsia, Actinomyces israelli and Chlamydia.

Examples of infectious fungi include: Cryptococcus neoformans,Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis,Chlamydia trachomatis and Candida albicans. Other infectious organisms(i.e., protists) include: Plasmodium such as Plasmodium falciparum,Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Toxoplasmagondii and Shistosoma.

Other medically relevant microorganisms have been described extensivelyin the literature, e.g., see C. G. A. Thomas, “Medical Microbiology”,Bailliere Tindall, Great Britain 1983, the entire contents of which ishereby incorporated by reference.

The compositions and methods of the invention are also useful fortreating cancer by stimulating an antigen-specific immune responseagainst a cancer antigen. A “tumor antigen” as used herein is acompound, such as a peptide, associated with a tumor or cancer and whichis capable of provoking an immune response. In particular, the compoundis capable of provoking an immune response when presented in the contextof an MHC molecule. Tumor antigens can be prepared from cancer cellseither by preparing crude extracts of cancer cells, for example, asdescribed in Cohen, et al., Cancer Research, 54:1055 (1994), bypartially purifying the antigens, by recombinant technology or by denovo synthesis of known antigens. Tumor antigens include antigens thatare antigenic portions of or are a whole tumor or cancer polypeptide.Such antigens can be isolated or prepared recombinantly or by any othermeans known in the art. Cancers or tumors include, but are not limitedto, biliary tract cancer; brain cancer; breast cancer; cervical cancer;choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer;gastric cancer; intraepithelial neoplasms; lymphomas; liver cancer; lungcancer (e.g. small cell and non-small cell); melanoma; neuroblastomas;oral cancer; ovarian cancer; pancreas cancer; prostate cancer; rectalcancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; andrenal cancer, as well as other carcinomas and sarcomas.

Antigenic determinant: As used herein, the term “antigenic determinant”is meant to refer to that portion of an antigen that is specificallyrecognized by either B- or T-lymphocytes. B-lymphocytes respond toforeign antigenic determinants via antibody production, whereasT-lymphocytes are the mediator of cellular immunity. Thus, antigenicdeterminants or epitopes are those parts of an antigen that arerecognized by antibodies, or in the context of an MHC, by T-cellreceptors.

Antigen presenting cell: As used herein, the term “antigen presentingcell” is meant to refer to a heterogenous population of leucocytes orbone marrow derived cells which possess an immunostimulatory capacity.For example, these cells are capable of generating peptides bound to MHCmolecules that can be recognized by T cells. The term is synonymous withthe term “accessory cell” and includes, for example, Langerhans' cells,interdigitating cells, B cells, macrophages and dendritic cells. Undersome conditions, epithelial cells, endothelial cells and other, non-bonemarrow derived cells may also serve as antigen presenting cells.

Association: As used herein, the term “association” as it applies to thefirst and second attachment sites, refers to the binding of the firstand second attachment sites that is preferably by way of at least onenon-peptide bond. The nature of the association may be covalent, ionic,hydrophobic, polar or any combination thereof, preferably the nature ofthe association is covalent, and again more preferably the associationis through at least one, preferably one, non-peptide bond. As usedherein, the term “association” as it applies to the first and secondattachment sites, not only encompass the direct binding or associationof the first and second attachment site forming the compositions of theinvention but also, alternatively and preferably, the indirectassociation or binding of the first and second attachment site leadingto the compositions of the invention, and hereby typically andpreferably by using a heterobifunctional cross-linker.

Attachment Site, First: As used herein, the phrase “first attachmentsite” refers to an element of non-natural or natural origin, typicallyand preferably being comprised by the virus-like particle, to which thesecond attachment site typically and preferably being comprised by theantigen or antigenic determinant may associate. The first attachmentsite may be a protein, a polypeptide, an amino acid, a peptide, a sugar,a polynucleotide, a natural or synthetic polymer, a secondary metaboliteor compound (biotin, fluorescein, retinol, digoxigenin, metal ions,phenylmethylsulfonylfluoride), or a combination thereof, or a chemicallyreactive group thereof. The first attachment site is located, typicallyand preferably on the surface, of the virus-like particle. Multiplefirst attachment sites are present on the surface of virus-like particletypically in a repetitive configuration. Preferably, the firstattachment site is a amino acid or a chemically reactive group thereof.

Attachment Site, Second: As used herein, the phrase “second attachmentsite” refers to an element associated with, typically and preferablybeing comprised by, the antigen or antigenic determinant to which thefirst attachment site located on the surface of the virus-like particlemay associate. The second attachment site of the antigen or antigenicdeterminant may be a protein, a polypeptide, a peptide, a sugar, apolynucleotide, a natural or synthetic polymer, a secondary metaboliteor compound (biotin, fluorescein, retinol, digoxigenin, metal ions,phenylmethylsulfonylfluoride), or a combination thereof, or a chemicallyreactive group thereof. At least one second attachment site is presenton the antigen or antigenic determinant. The term “antigen or antigenicdeterminant with at least one second attachment site” refers, therefore,to an antigen or antigenic construct comprising at least the antigen orantigenic determinant and the second attachment site. However, inparticular for a second attachment site, which is of non-natural origin,i.e. not naturally occurring within the antigen or antigenicdeterminant, these antigen or antigenic constructs comprise an “aminoacid linker”.

Bound: As used herein, the term “bound” refers to binding that may becovalent, e.g., by chemically coupling, or non-covalent, e.g., ionicinteractions, hydrophobic interactions, hydrogen bonds, etc. Covalentbonds can be, for example, ester, ether, phosphoester, amide, peptide,imide, carbon-sulfur bonds, carbon-phosphorus bonds, and the like. Theterm “bound” is broader than and includes terms such as “coupled”,“fused”, “associatede” and “attached”. Moreover, with respect to theimmunostimulatory substance being bound to the virus-like particle theterm “bound” also includes the enclosement, or partial enclosement, ofthe immunostimulatory substance. Therefore, with respect to theimmunostimulatory substance being bound to the virus-like particle theterm “bound” is broader than and includes terms such as “coupled,”“fused,” “enclosed”, “packaged” and “attached.” For example, theimmunostimulatory substance such as the unmethylated CpG-containingoligonucleotide can be enclosed by the VLP without the existence of anactual binding, neither covalently nor non-covalently.

Coat protein(s): As used herein, the term “coat protein(s)” refers tothe protein(s) of a bacteriophage or a RNA-phage capable of beingincorporated within the capsid assembly of the bacteriophage or theRNA-phage. However, when referring to the specific gene product of thecoat protein gene of RNA-phages the term “CP” is used. For example, thespecific gene product of the coat protein gene of RNA-phage Qβ isreferred to as “Qβ CP”, whereas the “coat proteins” of bacteriophage Qβcomprise the “Qβ CP” as well as the A1 protein. The capsid ofBacteriophage Qβ is composed mainly of the Qβ CP, with a minor contentof the A1 protein. Likewise, the VLP Qβ coat protein contains mainly QβCP, with a minor content of A1 protein.

Coupled: As used herein, the term “coupled” refers to attachment bycovalent bonds or by strong non-covalent interactions. With respect tothe coupling of the antigen to the virus-like particle the term“coupled” preferably refers to attachment by covalent bonds. Moreover,with respect to the coupling of the antigen to the virus-like particlethe term “coupled” preferably refers to association and attachment,respectively, by at least one non-peptide bond. Any method normally usedby those skilled in the art for the coupling of biologically activematerials can be used in the present invention.

Fusion: As used herein, the term “fusion” refers to the combination ofamino acid sequences of different origin in one polypeptide chain byin-frame combination of their coding nucleotide sequences. The term“fusion” explicitly encompasses internal fusions, i.e., insertion ofsequences of different origin within a polypeptide chain, in addition tofusion to one of its termini.

CpG: As used herein, the term “CpG” refers to an oligonucleotide whichcontains at least one unmethylated cytosine, guanine dinucleotidesequence (e.g. “CpG DNA” or DNA containing a cytosine followed byguanosine and linked by a phosphate bond) and stimulates/activates, e.g.has a mitogenic effect on, or induces or increases cytokine expressionby, a vertebrate cell. For example, CpGs can be useful in activating Bcells, NK cells and antigen-presenting cells, such as monocytes,dendritic cells and macrophages, and T cells. The CpGs can includenucleotide analogs such as analogs containing phosphorothioester bondsand can be double-stranded or single-stranded. Generally,double-stranded molecules are more stable in vivo, while single-strandedmolecules have increased immune activity.

Epitope: As used herein, the term “epitope” refers to portions of apolypeptide having antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably in a human. An “immunogenicepitope,” as used herein, is defined as a portion of a polypeptide thatelicits an antibody response or induces a T-cell response in an animal,as determined by any method known in the art. (See, for example, Geysenet al., Proc. Natl. Acad. Sci. USA 81:3998 4002 (1983)). The term“antigenic epitope,” as used herein, is defined as a portion of aprotein to which an antibody can immunospecifically bind its antigen asdetermined by any method well known in the art. Immunospecific bindingexcludes non specific binding but does not necessarily exclude crossreactivity with other antigens. Antigenic epitopes need not necessarilybe immunogenic. Antigenic epitopes can also be T-cell epitopes, in whichcase they can be bound immunospecifically by a T-cell receptor withinthe context of an MHC molecule.

An epitope can comprise 3 amino acids in a spatial conformation which isunique to the epitope. Generally, an epitope consists of at least about5 such amino acids, and more usually, consists of at least about 8-10such amino acids. If the epitope is an organic molecule, it may be assmall as Nitrophenyl.

Immune response: As used herein, the term “immune response” refers to ahumoral immune response and/or cellular immune response leading to theactivation or proliferation of B- and/or T-lymphocytes. In someinstances, however, the immune responses may be of low intensity andbecome detectable only when using at least one substance in accordancewith the invention. “Immunogenic” refers to an agent used to stimulatethe immune system of a living organism, so that one or more functions ofthe immune system are increased and directed towards the immunogenicagent. An “immunogenic polypeptide” is a polypeptide that elicits acellular and/or humoral immune response, whether alone or linked to acarrier in the presence or absence of an adjuvant.

Immunization: As used herein, the terms “immunize” or “immunization” orrelated terms refer to conferring the ability to mount a substantialimmune response (comprising antibodies or cellular immunity such aseffector CTL) against a target antigen or epitope. These terms do notrequire that complete immunity be created, but rather that an immuneresponse be produced which is substantially greater than baseline. Forexample, a mammal may be considered to be immunized against a targetantigen if the cellular and/or humoral immune response to the targetantigen occurs following the application of methods of the invention.

Immunostimulatory nucleic acid: As used herein, the termimmunostimulatory nucleic acid refers to a nucleic acid capable ofinducing and/or enhancing an immune response. Immunostimulatory nucleicacids, as used herein, comprise ribonucleic acids and in particulardeoxyribonucleic acids. Preferably, immunostimulatory nucleic acidscontain at least one CpG motif e.g. a CG dinucleotide in which the C isunmethylated. The CG dinucleotide can be part of a palindromic sequenceor can be encompassed within a non-palindromic sequence.Immunostimulatory nucleic acids not containing CpG motifs as describedabove encompass, by way of example, nucleic acids lacking CpGdinucleotides, as well as nucleic acids containing CG motifs with amethylated CG dinucleotide. The term “immunostimulatory nucleic acid” asused herein should also refer to nucleic acids that contain modifiedbases such as 4-bromo-cytosine.

Immunostimulatory substance: As used herein, the term “immunostimulatorysubstance” refers to a substance capable of inducing and/or enhancing animmune response. Immunostimulatory substances, as used herein, include,but are not limited to, toll-like receptor activing substances andsubstances inducing cytokine secretion. Toll-like receptor activatingsubstances include, but are not limited to, immunostimulatory nucleicacids, peptideoglycans, lipopolysaccharides, lipoteichonic acids,imidazoquinoline compounds, flagellins, lipoproteins, andimmunostimulatory organic substances such as taxol.

Natural origin: As used herein, the term “natural origin” means that thewhole or parts thereof are not synthetic and exist or are produced innature.

Non-natural: As used herein, the term generally means not from nature,more specifically, the term means from the hand of man.

Non-natural origin: As used herein, the term “non-natural origin”generally means synthetic or not from nature; more specifically, theterm means from the hand of man.

Ordered and repetitive antigen or antigenic determinant array: As usedherein, the term “ordered and repetitive antigen or antigenicdeterminant array” generally refers to a repeating pattern of antigen orantigenic determinant, characterized by a typically and preferablyuniform spacial arrangement of the antigens or antigenic determinantswith respect to the core particle and virus-like particle, respectively.In one embodiment of the invention, the repeating pattern may be ageometric pattern. Typical and preferred examples of suitable orderedand repetitive antigen or antigenic determinant arrays are those whichpossess strictly repetitive paracrystalline orders of antigens orantigenic determinants, preferably with spacings of 0.5 to 30nanometers, more preferably 3 to 15 nanometers, even more preferably 3to 8 nanometers.

Oligonucleotide: As used herein, the terms “oligonucleotide” or“oligomer” refer to a nucleic acid sequence comprising 2 or morenucleotides, generally at least about 6 nucleotides to about 100,000nucleotides, preferably about 6 to about 2000 nucleotides, and morepreferably about 6 to about 300 nucleotides, even more preferably about20 to about 300 nucleotides, and even more preferably about 20 to about100 nucleotides. The terms “oligonucleotide” or “oligomer” also refer toa nucleic acid sequence comprising more than 100 to about 2000nucleotides, preferably more than 100 to about 1000 nucleotides, andmore preferably more than 100 to about 500 nucleotides.“Oligonucleotide” also generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Oligonucleotide” includes, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “oligonucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. Further, an oligonucleotide can besynthetic, genomic or recombinant, e.g., λ-DNA, cosmid DNA, artificialbacterial chromosome, yeast artificial chromosome and filamentous phagesuch as M13. In a very preferred embodiment of the present invention,the oligonucleotide is a synthetic oligonucleotide.

The term “oligonucleotide” also includes DNAs or RNAs containing one ormore modified bases and DNAs or RNAs with backbones modified forstability or for other reasons. For example, suitable nucleotidemodifications/analogs include peptide nucleic acid, inosin, tritylatedbases, phosphorothioates, alkylphosphorothioates, 5-nitroindoledeoxyribofuranosyl, 5-methyldeoxycytosine and5,6-dihydro-5,6-dihydroxydeoxythymidine. A variety of modifications havebeen made to DNA and RNA; thus, “oligonucleotide” embraces chemically,enzymatically or metabolically modified forms of polynucleotides astypically found in nature, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells. Other nucleotideanalogs/modifications will be evident to those skilled in the art.

Packaged: The term “packaged” as used herein refers to the state of animmunostimulatory substance, preferably of an immunostimulatory nucleicacid in relation to the VLP. The term “packaged” as used herein includesbinding that may be covalent, e.g., by chemically coupling, ornon-covalent, e.g., ionic interactions, hydrophobic interactions,hydrogen bonds, etc. Covalent bonds can be, for example, ester, ether,phosphoester, amide, peptide, imide, carbon-sulfur bonds such asthioether bonds, carbon-phosphorus bonds, and the like. The term alsoincludes the enclosement, or partial enclosement, of a substance. Theterm “packaged” includes terms such as “coupled, “enclosed” and“attached.” For example, the immunostimulatory substance such as theunmethylated CpG-containing oligonucleotide can be enclosed by the VLPwithout the existence of an actual binding, neither covalently nornon-covalently. In preferred embodiments, in particular, ifimmunostimulatory nucleic acids are the immunostimulatory substances,the term “packaged” indicates that the immunostimulatory nucleic acid ina packaged state is not accessible to DNAse or RNAse hydrolysis. Inpreferred embodiments, the immunostimulatory nucleic acid is packagedinside the VLP capsids, most preferably in a non-covalent manner.

The compositions of the invention can be combined, optionally, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human or other animal. Theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application.

Organic molecule: As used herein, the term “organic molecule” refers toany chemical entity of natural or synthetic origin. In particular theterm “organic molecule” as used herein encompasses, for example, anymolecule being a member of the group of nucleotides, lipids,carbohydrates, polysaccharides, lipopolysaccharides, steroids,alkaloids, terpenes and fatty acids, being either of natural orsynthetic origin. In particular, the term “organic molecule” encompassesmolecules such as nicotine, cocaine, heroin or other pharmacologicallyactive molecules contained in drugs of abuse. In general an organicmolecule contains or is modified to contain a chemical functionalityallowing its coupling, binding or other method of attachment to thevirus-like particle in accordance with the invention.

Polypeptide: As used herein, the term “polypeptide” refers to a moleculecomposed of monomers (amino acids) linearly linked by amide bonds (alsoknown as peptide bonds). It indicates a molecular chain of amino acidsand does not refer to a specific length of the product. Thus, peptides,oligopeptides and proteins are included within the definition ofpolypeptide. This term is also intended to refer to post-expressionmodifications of the polypeptide, for example, glycosolations,acetylations, phosphorylations, and the like. A recombinant or derivedpolypeptide is not necessarily translated from a designated nucleic acidsequence. It may also be generated in any manner, including chemicalsynthesis.

A substance which “enhances” an immune response refers to a substance inwhich an immune response is observed that is greater or intensified ordeviated in any way with the addition of the substance when compared tothe same immune response measured without the addition of the substance.For example, the lytic activity of cytotoxic T cells can be measured,e.g. using a 51Cr release assay, typically and preferably as outlined inCurrent Protocols in Immunology, Editors: John E. Coligan et al.; JohnWiley & Sons Inc., with and without the substance. The amount of thesubstance at which the CTL lytic activity is enhanced as compared to theCTL lytic activity without the substance is said to be an amountsufficient to enhance the immune response of the animal to the antigen.In a preferred embodiment, the immune response in enhanced by a factorof at least about 2, more preferably by a factor of about 3 or more. Theamount of cytokines secreted may also be altered.

Effective Amount: As used herein, the term “effective amount” refers toan amount necessary or sufficient to realize a desired biologic effect.An effective amount of the composition would be the amount that achievesthis selected result, and such an amount could be determined as a matterof routine by a person skilled in the art. For example, an effectiveamount for treating an immune system deficiency could be that amountnecessary to cause activation of the immune system, resulting in thedevelopment of an antigen specific immune response upon exposure toantigen. The term is also synonymous with “sufficient amount.”

The effective amount for any particular application can vary dependingon such factors as the disease or condition being treated, theparticular composition being administered, the size of the subject,and/or the severity of the disease or condition. One of ordinary skillin the art can empirically determine the effective amount of aparticular composition of the present invention without necessitatingundue experimentation.

Self antigen: As used herein, the term “self antigen” refers to proteinsencoded by the host's genome or DNA and products generated by proteinsor RNA encoded by the host's genome or DNA are defined as self.Preferably, the term “self antigen”, as used herein, refers to proteinsencoded by the human genome or DNA and products generated by proteins orRNA encoded by the human genome or DNA are defined as self. Theinventive compositions, pharmaceutical compositions and vaccinescomprising self antigens are in particular capable of breaking toleranceagainst a self antigen when applied to the host. In this context,“breaking tolerance against a self antigen” shall refer to enhancing animmune response, as defined herein, and preferably enhancing a B or a Tcell response, specific for the self antigen when applying the inventivecompositions, pharmaceutical compositions and vaccines comprising theself antigen to the host. In addition, proteins that result from acombination of two or several self-molecules or that represent afraction of a self-molecule and proteins that have a high homology twoself-molecules as defined above (>95%, preferably >97%, morepreferably >99%) may also be considered self. In a further preferredembodiment of the present invention, the antigen is a self antigen. Verypreferred embodiments of self-antigens useful for the present inventionare described WO 02/056905, the disclosures of which are herewithincorporated by reference in its entirety.

Treatment: As used herein, the terms “treatment”, “treat”, “treated” or“treating” refer to prophylaxis and/or therapy. When used with respectto an infectious disease, for example, the term refers to a prophylactictreatment which increases the resistance of a subject to infection witha pathogen or, in other words, decreases the likelihood that the subjectwill become infected with the pathogen or will show signs of illnessattributable to the infection, as well as a treatment after the subjecthas become infected in order to fight the infection, e.g., reduce oreliminate the infection or prevent it from becoming worse.

Vaccine: As used herein, the term “vaccine” refers to a formulationwhich contains the composition of the present invention and which is ina form that is capable of being administered to an animal. Typically,the vaccine comprises a conventional saline or buffered aqueous solutionmedium in which the composition of the present invention is suspended ordissolved. In this form, the composition of the present invention can beused conveniently to prevent, ameliorate, or otherwise treat acondition. Upon introduction into a host, the vaccine is able to provokean immune response including, but not limited to, the production ofantibodies, cytokines and/or the activation of cytotoxic T cells,antigen presenting cells, helper T cells, dendritic cells and/or othercellular responses.

Optionally, the vaccine of the present invention additionally includesan adjuvant which can be present in either a minor or major proportionrelative to the compound of the present invention. The term “adjuvant”as used herein refers to non-specific stimulators of the immune responseor substances that allow generation of a depot in the host which whencombined with the vaccine of the present invention provide for an evenmore enhanced immune response. A variety of adjuvants can be used.Examples include incomplete Freund's adjuvant, aluminum hydroxide andmodified muramyldipeptide. The term “adjuvant” as used herein alsorefers to typically specific stimulators of the immune response whichwhen combined with the vaccine of the present invention provide for aneven more enhanced and typically specific immune response. Examplesinclude, but limited to, GM-CSF, IL-2, IL-12, IFNα. Further examples arewithin the knowledge of the person skilled in the art.

Virus-like particle: As used herein, the term “virus-like particle”refers to a structure resembling a virus particle but which has not beendemonstrated to be pathogenic. Typically, a virus-like particle inaccordance with the invention does not carry genetic informationencoding for the proteins of the virus-like particle. In general,virus-like particles lack the viral genome and, therefore, arenoninfectious. Also, virus-like particles can often be produced in largequantifies by heterologous expression and can be easily purified. Somevirus-like particles may contain nucleic acid distinct from theirgenome. As indicated, a virus-like particle in accordance with theinvention is non replicative and noninfectious since it lacks all orpart of the viral genome, in particular the replicative and infectiouscomponents of the viral genome. A virus-like particle in accordance withthe invention may contain nucleic acid distinct from their genome. Atypical and preferred embodiment of a virus-like particle in accordancewith the present invention is a viral capsid such as the viral capsid ofthe corresponding virus, bacteriophage, or RNA-phage. The terms “viralcapsid” or “capsid”, as interchangeably used herein, refer to amacromolecular assembly composed of viral protein subunits. Typicallyand preferably, the viral protein subunits assemble into a viral capsidand capsid, respectively, having a structure with an inherent repetitiveorganization, wherein said structure is, typically, spherical ortubular. For example, the capsids of RNA-phages or HBcAg's have aspherical form of icosahedral symmetry. The term “capsid-like structure”as used herein, refers to a macromolecular assembly composed of viralprotein subunits ressembling the capsid morphology in the above definedsense but deviating from the typical symmetrical assembly whilemaintaining a sufficient degree of order and repetitiveness.

Virus-like particle of a bacteriophage: As used herein, the term“virus-like particle of a bacteriophage” refers to a virus-like particleresembling the structure of a bacteriophage, being non replicative andnoninfectious, and lacking at least the gene or genes encoding for thereplication machinery of the bacteriophage, and typically also lackingthe gene or genes encoding the protein or proteins responsible for viralattachment to or entry into the host. This definition should, however,also encompass virus-like particles of bacteriophages, in which theaforementioned gene or genes are still present but inactive, and,therefore, also leading to non-replicative and noninfectious virus-likeparticles of a bacteriophage.

VLP of RNA phage coat protein: The capsid structure formed from theself-assembly of 180 subunits of RNA phage coat protein and optionallycontaining host RNA is referred to as a “VLP of RNA phage coat protein”.A specific example is the VLP of Qβ coat protein. In this particularcase, the VLP of Qβ coat protein may either be assembled exclusivelyfrom Qβ CP subunits (SEQ ID: No 10) generated by expression of a Qβ CPgene containing, for example, a TAA stop codon precluding any expressionof the longer A1 protein through suppression, see Kozlovska, T. M., etal., Intervirology 39: 9-15 (1996)), or additionally contain A1 proteinsubunits (SEQ ID: No 11) in the capsid assembly. The readthrough processhas a low efficiency and is leading to an only very low amount of A1protein in the VLPs. An extensive number of examples have been performedwith different combinations of ISS packaged and antigen coupled. Nodifferences in the coupling efficiency and the packaging have beenobserved when VLPs of Qβ coat protein assembled exclusively from Qβ CPsubunits or VLPs of Qβ coat protein containing additionally A1 proteinsubunits in the capsids were used. Furthermore, no difference of theimmune response between these QβVLP preparations was observed.Therefore, for the sake of clarity the term “QβVLP” is used throughoutthe description of the examples either for VLPs of Qβ coat proteinassembled exclusively from Qβ CP subunits or VLPs of Qβ coat proteincontaining additionally A1 protein subunits in the capsids.

The term “virus particle” as used herein refers to the morphologicalform of a virus. In some virus types it comprises a genome surrounded bya protein capsid; others have additional structures (e.g., envelopes,tails, etc.).

Non-enveloped viral particles are made up of a proteinaceous capsid thatsurrounds and protects the viral genome. Enveloped viruses also have acapsid structure surrounding the genetic material of the virus but, inaddition, have a lipid bilayer envelope that surrounds the capsid. In apreferred embodiment of the invention, the VLP's are free of alipoprotein envelope or a lipoprotein-containing envelope. In a furtherpreferred embodiment, the VLP's are free of an envelope altogether.

One, a, or an: When the terms “one,” “a,” or “an” are used in thisdisclosure, they mean “at least one” or “one or more,” unless otherwiseindicated.

As will be clear to those skilled in the art, certain embodiments of theinvention involve the use of recombinant nucleic acid technologies suchas cloning, polymerase chain reaction, the purification of DNA and RNA,the expression of recombinant proteins in prokaryotic and eukaryoticcells, etc. Such methodologies are well known to those skilled in theart and can be conveniently found in published laboratory methodsmanuals (e.g., Sambrook, J. et al., eds., MOLECULAR CLONING, ALABORATORY MANUAL, 2nd. edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al., eds., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997)).Fundamental laboratory techniques for working with tissue culture celllines (Celis, J., ed., CELL BIOLOGY, Academic Press, 2nd edition,(1998)) and antibody-based technologies (Harlow, E. and Lane, D.,“Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1988); Deutscher, M. P., “Guide to ProteinPurification,” Meth. Enzymol. 128, Academic Press San Diego (1990);Scopes, R. K., “Protein Purification Principles and Practice,” 3rd ed.,Springer-Verlag, New York (1994)) are also adequately described in theliterature, all of which are incorporated herein by reference.

2. Compositions and Methods for Enhancing an Immune Response

The disclosed invention provides compositions and methods for enhancingan immune response against one or more antigens in an animal.Compositions of the invention comprise, or alternatively consistessentially of, or alternatively consist of, a virus-like particle andat least one immunostimulatory substance, wherein the immunostimulatorysubstance is bound to said virus-like particle, and wherein theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence, andwherein said palindromic sequence is flanked at its 3′-terminus and atits 5′-terminus by less than 10 guanosine entities. Furthermore, theinvention conveniently enables the practitioner to construct such acomposition for various treatment and/or prophylactic preventionpurposes, which include the prevention and/or treatment of infectiousdiseases, as well as chronic infectious diseases, and the preventionand/or treatment of cancers, for example.

Virus-like particles in the context of the present application refer tostructures resembling a virus particle but which are not pathogenic. Ingeneral, virus-like particles lack the viral genome and, therefore, arenoninfectious. Also, virus-like particles can be produced in largequantities by heterologous expression and can be easily purified.

In a preferred embodiment, the virus-like particle is a recombinantvirus-like particle. The skilled artisan can produce VLPs usingrecombinant DNA technology and virus coding sequences which are readilyavailable to the public. For example, the coding sequence of a virusenvelope or core protein can be engineered for expression in abaculovirus expression vector using a commercially available baculovirusvector, under the regulatory control of a virus promoter, withappropriate modifications of the sequence to allow functional linkage ofthe coding sequence to the regulatory sequence. The coding sequence of avirus envelope or core protein can also be engineered for expression ina bacterial expression vector, for example.

Examples of VLPs include, but are not limited to, the capsid proteins ofHepatitis B virus, measles virus, Sindbis virus, rotavirus,foot-and-mouth-disease virus, Norwalk virus, the retroviral GAG protein,the retrotransposon Ty protein p1, the surface protein of Hepatitis Bvirus, human papilloma virus, human polyoma virus, BK virus (BKV), RNAphages, Ty, fr-phage, GA-phage, AP 205-phage and, in particular,Qβ-phage.

As will be readily apparent to those skilled in the art, the VLP of theinvention is not limited to any specific form. The particle can besynthesized chemically or through a biological process, which can benatural or non-natural. By way of example, this type of embodimentincludes a virus-like particle or a recombinant form thereof.

In a more specific embodiment, the VLP can comprise, or alternativelyconsist of, recombinant polypeptides of Rotavirus; recombinantpolypeptides of Norwalk virus; recombinant polypeptides of Alphavirus;recombinant proteins which form bacterial pili or pilus like structures;recombinant polypeptides of Foot and Mouth Disease virus; recombinantpolypeptides of measles virus, recombinant polypeptides of Sindbisvirus, recombinant polypeptides of Retrovirus; recombinant polypeptidesof Hepatitis B virus (e.g., a HBcAg); recombinant polypeptides ofTobacco mosaic virus; recombinant polypeptides of Flock House Virus;recombinant polypeptides of human Papillomavirus; recombinantpolypeptides of Polyoma virus and, in particular, recombinantpolypeptides of human Polyoma virus, and in particular recombinantpolypeptides of BK virus; recombinant polypeptides of bacteriophages,recombinant polypeptides of RNA phages; recombinant polypeptides of Ty;recombinant polypeptides of fr-phage, recombinant polypeptides ofGA-phage, recombinant polypeptides of AP 205-phage and, in particular,recombinant polypeptides of Qβ-phage. The virus-like particle canfurther comprise, or alternatively consist of, one or more fragments ofsuch polypeptides, as well as variants of such polypeptides. Variants ofpolypeptides can share, for example, at least 80%, 85%, 90%, 95%, 97%,or 99% identity at the amino acid level with their wild typecounterparts.

In a preferred embodiment, the virus-like particle comprises, consistsessentially of, or alternatively consists of recombinant proteins, orfragments thereof, of a RNA-phage. Preferably, the RNA-phage is selectedfrom the group consisting of a) bacteriophage Qβ; b) bacteriophage R17;c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f)bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i)bacteriophage NL95; k) bacteriophage f2; l) bacteriophage PP7; and m)bacteriophage AP205.

In another preferred embodiment of the present invention, the virus-likeparticle comprises, or alternatively consists essentially of, oralternatively consists of recombinant proteins, or fragments thereof, ofthe RNA-bacteriophage Qβ or of the RNA-bacteriophage fr or of theRNA-bacteriophage AP205.

In a further preferred embodiment of the present invention, therecombinant proteins comprise, or alternatively consist essentially of,or alternatively consist of coat proteins of RNA phages.

RNA-phage coat proteins forming capsids or VLPs, or fragments of thebacteriophage coat proteins compatible with self-assembly into a capsidor a VLP, are, therefore, further preferred embodiments of the presentinvention. Bacteriophage Qβ coat proteins, for example, can be expressedrecombinantly in E. coli. Further, upon such expression these proteinsspontaneously form capsids. Additionally, these capsids form a structurewith an inherent repetitive organization.

Specific preferred examples of bacteriophage coat proteins which can beused to prepare compositions of the invention include the coat proteinsof RNA bacteriophages such as bacteriophage Qβ (SEQ ID NO:10; PIRDatabase, Accession No. VCBPQβ referring to Qβ CP and SEQ ID NO: 11;Accession No. AAA16663 referring to Qβ A1 protein), bacteriophage R17(PIR Accession No. VCBPR7), bacteriophage fr (SEQ ID NO:13; PIRAccession No. VCBPFR), bacteriophage GA (SEQ ID NO:14; GenBank AccessionNo. NP-040754), bacteriophage SP (GenBank Accession No. CAA30374referring to SP CP and Accession No. NP_(—)695026 referring to SP A1protein), bacteriophage MS2 (PIR Accession No. VCBPM2), bacteriophageM11 (GenBank Accession No. AAC06250), bacteriophage MX1 (GenBankAccession No. AAC14699), bacteriophage NL95 (GenBank Accession No.AAC14704), bacteriophage f2 (GenBank Accession No. P03611),bacteriophage PP7 (SEQ ID NO: 22), and bacteriophage AP205 (SEQ ID NO:31). Furthermore, the A1 protein of bacteriophage Qβ or C-terminaltruncated forms missing as much as 100, 150 or 180 amino acids from itsC-terminus may be incorporated in a capsid assembly of Qβ coat proteins.Generally, the percentage of QβA1 protein relative to Qβ CP in thecapsid assembly will be limited, in order to ensure capsid formation.Further specific examples of bacteriophage coat proteins are describedin WO 02/056905 on page 45 and 46 incorporated herein by reference.Further preferred virus-like particles of RNA-phages, in particular ofQβ in accordance of this invention are disclosed in WO 02/056905, thedisclosure of which is herewith incorporated by reference in itsentirety.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively consists essentially of,or alternatively consists of recombinant proteins, or fragments thereof,of a RNA-phage, wherein the recombinant proteins comprise, consistessentially of or alternatively consist of mutant coat proteins of a RNAphage, preferably of mutant coat proteins of the RNA phages mentionedabove. In another preferred embodiment, the mutant coat proteins of theRNA phage have been modified by removal of at least one lysine residueby way of substitution, or by addition of at least one lysine residue byway of substitution; alternatively, the mutant coat proteins of the RNAphage have been modified by deletion of at least one lysine residue, orby addition of at least one lysine residue by way of insertion. Thedeletion, substitution or addition of at least one lysine residue allowsvarying the degree of coupling, i.e. the amount of antigens per subunitsof the VLP of the RNA-phages, in particular, to match and tailor therequirements of the vaccine. In a preferred embodiment of the presentinvention, on average at least 1.0 antigen peptide per subunit arelinked to the VLP of the RNA-phage. This value is calculated as anaverage over all the subunits or monomers of the VLP of the RNA-phage.In a further preferred embodiment of the present invention, at least1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or at least 2.0 antigenpolypeptides are linked to the VLP of the RNA-phages as being calculatedas a coupling average over all the subunits or monomers of the VLP ofthe RNA-phage.

In another preferred embodiment, the virus-like particle comprises, oralternatively consists essentially of, or alternatively consists ofrecombinant proteins, or fragments thereof, of the RNA-bacteriophage Qβ,wherein the recombinant proteins comprise, or alternatively consistessentially of, or alternatively consist of coat proteins having anamino acid sequence of SEQ ID NO:10, or a mixture of coat proteinshaving amino acid sequences of SEQ ID NO:10 and of SEQ ID NO: 11 ormutants of SEQ ID NO: 11 and wherein the N-terminal methionine ispreferably cleaved.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, consists essentially of or alternativelyconsists of recombinant proteins of Qβ, or fragments thereof, whereinthe recombinant proteins comprise, or alternatively consist essentiallyof, or alternatively consist of mutant Qβ coat proteins. In anotherpreferred embodiment, these mutant coat proteins have been modified byremoval of at least one lysine residue by way of substitution, or byaddition of at least one lysine residue by way of substitution.Alternatively, these mutant coat proteins have been modified by deletionof at least one lysine residue, or by addition of at least one lysineresidue by way of insertion.

Four lysine residues are exposed on the surface of the capsid of Qβ coatprotein. Qβ mutants, for which exposed lysine residues are replaced byarginines can also be used for the present invention. The following Qβcoat protein mutants and mutant Qβ VLPs can, thus, be used in thepractice of the invention: “Qβ-240” (Lys13-Arg; SEQ ID NO:20), “Qβ-243”(Asn 10-Lys; SEQ ID NO:21), “Qβ-250” (Lys 2-Arg, Lys13-Arg; SEQ IDNO:22), “Qβ-251” (SEQ ID NO:23) and “Qβ-259” (Lys 2-Arg, Lys16-Arg; SEQID NO:24). Thus, in further preferred embodiment of the presentinvention, the virus-like particle comprises, consists essentially of oralternatively consists of recombinant proteins of mutant Qβ coatproteins, which comprise proteins having an amino acid sequence selectedfrom the group of a) the amino acid sequence of SEQ ID NO: 20; b) theamino acid sequence of SEQ ID NO:21; c) the amino acid sequence of SEQID NO: 22; d) the amino acid sequence of SEQ ID NO:23; and e) the aminoacid sequence of SEQ ID NO: 24. The construction, expression andpurification of the above indicated Qβ coat proteins, mutant Qβ coatprotein VLPs and capsids, respectively, are disclosed in WO02/056905. Inparticular is hereby referred to Example 18 of above mentionedapplication.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively consists essentially of,or alternatively consists of recombinant proteins of Qβ, or fragmentsthereof, wherein the recombinant proteins comprise, consist essentiallyof or alternatively consist of a mixture of either one of the foregoingQβ mutants and the corresponding A1 protein.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant proteins, or fragments thereof,of RNA-phage AP205.

The AP205 genome consists of a maturation protein, a coat protein, areplicase and two open reading frames not present in related phages; alysis gene and an open reading frame playing a role in the translationof the maturation gene (Klovins, J., et al., J. Gen. Virol. 83: 1523-33(2002)). AP205 coat protein can be expressed from plasmid pAP283-58 (SEQID NO: 30), which is a derivative of pQb10 (Kozlovska, T. M. et al.,Gene 137:133-37 (1993)), and which contains an AP205 ribosomal bindingsite. Alternatively, AP205 coat protein may be cloned into pQb185,downstream of the ribosomal binding site present in the vector. Bothapproaches lead to expression of the protein and formation of capsids asdescribed in WO 04/007538 which is incorporated by reference in itsentirety. Vectors pQb10 and pQb185 are vectors derived from pGEM vector,and expression of the cloned genes in these vectors is controlled by thetrp promoter (Kozlovska, T. M. et al., Gene 137:133-37 (1993)). PlasmidpAP283-58 (SEQ ID NO:30) comprises a putative AP205 ribosomal bindingsite in the following sequence, which is downstream of the XbaI site,and immediately upstream of the ATG start codon of the AP205 coatprotein: tctagaATTTTCTGCGCACCCAT CCCGGGTGGCGCCCAAAGTGAGGAAAATCACatg(bases 77-133 of SEQ ID NO: 30). The vector pQb185 comprises a ShineDelagamo sequence downstream from the XbaI site and upstream of thestart codon (tctagaTTAACCCAACGCGTAGGAGTCAGGCCatg (SEQ ID NO: 46), ShineDelagarno sequence underlined).

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant coat proteins, or fragmentsthereof, of the RNA-phage AP205.

This preferred embodiment of the present invention, thus, comprisesAP205 coat proteins that form capsids. Such proteins are recombinantlyexpressed, or prepared from natural sources. AP205 coat proteinsproduced in bacteria spontaneously form capsids, as evidenced byElectron Microscopy (EM) and immunodiffusion. The structural propertiesof the capsid formed by the AP205 coat protein (SEQ ID NO: 31) and thoseformed by the coat protein of the AP205 RNA phage are nearlyindistinguishable when seen in EM. AP205 VLPs are highly immunogenic,and can be linked with antigens and/or antigenic determinants togenerate vaccine constructs displaying the antigens and/or antigenicdeterminants oriented in a repetitive manner. High titers are elicitedagainst the so displayed antigens showing that bound antigens and/orantigenic determinants are accessible for interacting with antibodymolecules and are immunogenic.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant mutant coat proteins, orfragments thereof, of the RNA-phage AP205.

Assembly-competent mutant forms of AP205 VLPs, including AP205 coatprotein with the subsitution of proline at amino acid 5 to threonine(SEQ ID NO: 32), may also be used in the practice of the invention andleads to a further preferred embodiment of the invention. These VLPs,AP205 VLPs derived from natural sources, or AP205 viral particles, maybe bound to antigens to produce ordered repetitive arrays of theantigens in accordance with the present invention.

AP205 P5-T mutant coat protein can be expressed from plasmid pAP281-32(SEQ ID No. 33), which is derived directly from pQb185, and whichcontains the mutant AP205 coat protein gene instead of the Qβ coatprotein gene. Vectors for expression of the AP205 coat protein aretransfected into E. coli for expression of the AP205 coat protein.

Methods for expression of the coat protein and the mutant coat protein,respectively, leading to self-assembly into VLPs are described in WO04/007538 which is incorporated by reference in its entirety. SuitableE. coli strains include, but are not limited to, E. coli K802, JM 109,RR1. Suitable vectors and strains and combinations thereof can beidentified by testing expression of the coat protein and mutant coatprotein, respectively, by SDS-PAGE and capsid formation and assembly byoptionally first purifying the capsids by gel filtration andsubsequently testing them in an immunodiffusion assay (Ouchterlony test)or Electron Microscopy (Kozlovska, T. M. et al., Gene 137:133-37(1993)).

AP205 coat proteins expressed from the vectors pAP283-58 and pAP281-32may be devoid of the initial Methionine amino-acid, due to processing inthe cytoplasm of E. coli. Cleaved, uncleaved forms of AP205 VLP, ormixtures thereof are further preferred embodiments of the invention.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of a mixture of recombinant coat proteins, orfragments thereof, of the RNA-phage AP205 and of recombinant mutant coatproteins, or fragments thereof, of the RNA-phage AP205.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of fragments of recombinant coat proteins orrecombinant mutant coat proteins of the RNA-phage AP205.

Recombinant AP205 coat protein fragments capable of assembling into aVLP and a capsid, respectively are also useful in the practice of theinvention. These fragments may be generated by deletion, eitherinternally or at the termini of the coat protein and mutant coatprotein, respectively. Insertions in the coat protein and mutant coatprotein sequence or fusions of antigen sequences to the coat protein andmutant coat protein sequence, and compatible with assembly into a VLP,are further embodiments of the invention and lead to chimeric AP205 coatproteins, and particles, respectively. The outcome of insertions,deletions and fusions to the coat protein sequence and whether it iscompatible with assembly into a VLP can be determined by electronmicroscopy.

The particles formed by the AP205 coat protein, coat protein fragmentsand chimeric coat proteins described above, can be isolated in pure formby a combination of fractionation steps by precipitation and ofpurification steps by gel filtration using e.g. Sepharose CL-4B,Sepharose CL-2B, Sepharose CL-6B columns and combinations thereof asdescribed in WO 04/007538 which is incorporated by reference in itsentirety. Other methods of isolating virus-like particles are known inthe art, and may be used to isolate the virus-like particles (VLPs) ofbacteriophage AP205. For example, the use of ultracentrifugation toisolate VLPs of the yeast retrotransposon Ty is described in U.S. Pat.No. 4,918,166, which is incorporated by reference herein in itsentirety.

The crystal structure of several RNA bacteriophages has been determined(Golmohammadi, R. et al., Structure 4:543-554 (1996)). Using suchinformation, one skilled in the art could readily identify surfaceexposed residues and modify bacteriophage coat proteins such that one ormore reactive amino acid residues can be inserted. Thus, one skilled inthe art could readily generate and identify modified forms ofbacteriophage coat proteins which can be used in the practice of theinvention. Thus, variants of proteins which form capsids or capsid-likestructures (e.g., coat proteins of bacteriophage Qβ, bacteriophage R17,bacteriophage fr, bacteriophage GA, bacteriophage SP, and bacteriophageMS2) can also be used for the inventive compositions and vaccinecompositions. Further possible examples of modified RNA bacteriophagesas well as variants of proteins and N- and C terminal truncation mutantswhich form capsids or capsid like structures, as well as methods forpreparing such compositions and vaccine compositions, respectively,which are suitable for use in the present invention are described in WO02/056905 on page 50, line 33 to page 52, line 29.

The invention thus includes compositions and vaccine compositionsprepared from proteins which form capsids or VLPs, methods for preparingthese compositions from individual protein subunits and VLPs or capsids,methods for preparing these individual protein subunits, nucleic acidmolecules which encode these subunits, and methods for vaccinatingand/or eliciting immunological responses in individuals using thesecompositions of the present invention.

In another preferred embodiment of the invention, the VLP's are free ofa lipoprotein envelope or a lipoprotein-containing envelope. In afurther preferred embodiment, the VLP's are free of an envelopealtogether.

The lack of a lipoprotein envelope or lipoprotein-containing envelopeand, in particular, the complete lack of an envelope leads to a moredefined virus-like particle in its structure and composition. Such moredefined virus-like particles, therefore, may minimize side-effects.Moreover, the lack of a lipoprotein-containing envelope or, inparticular, the complete lack of an envelope avoids or minimizesincorporation of potentially toxic molecules and pyrogens within thevirus-like particle.

In one embodiment, the invention provides a vaccine composition of theinvention comprising a virus-like particle, wherein preferably saidvirus-like particle is a recombinant virus-like particle. Preferably,the virus-like particle comprises, or alternatively consist essentiallyof, or alternatively consists of, recombinant proteins, or fragmentsthereof, of a RNA-phage, preferably of coat proteins of RNA phages.Alternatively, the recombinant proteins of the virus-like particle ofthe vaccine composition of the invention comprise, or alternativelyconsist essentially of, or alternatively consist of mutant coat proteinsof RNA phages, wherein the RNA-phage is selected from the groupconsisting of: (a) bacteriophage Qβ; (b) bacteriophage R17; (c)bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP; (f)bacteriophage MS2; (g) bacteriophage M11; (h) bacteriophage MX1; (i)bacteriophage NL95; (k) bacteriophage f2; (l) bacteriophage PP7; and (m)bacteriophage AP205.

In a preferred embodiment, the mutant coat proteins of said RNA phagehave been modified by removal, or by addition of at least one lysineresidue by way of substitution. In another preferred embodiment, themutant coat proteins of said RNA phage have been modified by deletion ofat least one lysine residue or by addition of at least one lysineresidue by way of insertion. In a preferred embodiment, the virus-likeparticle comprises recombinant proteins or fragments thereof, ofRNA-phage Qβ or alternatively of RNA-phage fr, or of RNA-phage AP205.

As previously stated, the invention includes virus-like particles orrecombinant forms thereof. Skilled artisans have the knowledge toproduce such particles and attach antigens thereto. Further preferredembodiments of the present invention hereto are disclosed in the ExampleSection.

In one embodiment, the virus-like particle comprises, or alternativelyconsists essentially of, or alternatively consists of recombinantproteins, or fragments thereof, of the BK virus (BKV), wherein therecombinant proteins comprise, or alternatively consist essentially of,or alternatively consist of proteins having an amino acid sequence ofSEQ ID NO:12. BK virus (BKV) is a non-enveloped double stranded DNAvirus belonging to the polyoma virus subfamily of the papovaviridae. VP1is the major capsid protein of BKV. VP1 has 362 amino acids (SEQ ID NO:12, Gene Bank entry: AAA46882) and is 42 kDa in size. When produced inE. coli, insect cells or yeast VP1 spontaneously forms capsid structures(Salunke D. M., et al., Cell 46(6):895-904 (1986); Sasnauskas, K., etal., Biol. Chem. 380(3):381-6 (1999); Sasnauskas, K., et al., 3rdInternational Workshop “Virus-like particles as vaccines” Berlin, Sep.26-29, 2001; Touze, A., et al., J Gen Virol. 82(Pt 12):3005-9 (2001).The capsid is organized in 72 VP1 pentamers forming an icosahedralstructure. The capsids have a diameter of approximately 45 nm.

In one embodiment, the particles used in compositions of the inventionare composed of a Hepatitis B capsid (core) protein (HBcAg) or afragment of a HBcAg which has been modified to either eliminate orreduce the number of free cysteine residues. Zhou et al. (J. Virol.66:5393 5398 (1992)) demonstrated that HBcAgs which have been modifiedto remove the naturally resident cysteine residues retain the ability toassociate and form multimeric structures. Thus, core particles suitablefor use in compositions of the invention include those comprisingmodified HBcAgs, or fragments thereof, in which one or more of thenaturally resident cysteine residues have been either deleted orsubstituted with another amino acid residue (e.g., a serine residue).

The HBcAg is a protein generated by the processing of a Hepatitis B coreantigen precursor protein. A number of isotypes of the HBcAg have beenidentified and their amino acids sequences are readily available tothose skilled in the art. For example, the HBcAg protein having theamino acid sequence shown in SEQ ID NO: 16 is 185 amino acids in lengthand is generated by the processing of a 212 amino acid Hepatitis B coreantigen precursor protein. This processing results in the removal of 29amino acids from the N terminus of the Hepatitis B core antigenprecursor protein. Similarly, the HBcAg protein that is 185 amino acidsin length is generated by the processing of a 214 amino acid Hepatitis Bcore antigen precursor protein.

In preferred embodiments, vaccine compositions of the invention will beprepared using the processed form of a HBcAg (i.e., a HBcAg from whichthe N terminal leader sequence of the Hepatitis B core antigen precursorprotein have been removed).

Further, when HBcAgs are produced under conditions where processing willnot occur, the HBcAgs will generally be expressed in “processed” form.For example, bacterial systems, such as E. coli, generally do not removethe leader sequences, also referred to as “signal peptides,” of proteinswhich are normally expressed in eukaryotic cells. Thus, when an E. coliexpression system directing expression of the protein to the cytoplasmis used to produce HBcAgs of the invention, these proteins willgenerally be expressed such that the N terminal leader sequence of theHepatitis B core antigen precursor protein is not present.

The preparation of Hepatitis B virus-like particles, which can be usedfor the present invention, is disclosed, for example, in WO 00/32227,and hereby in particular in Examples 17 to 19 and 21 to 24, as well asin WO 01/85208, and hereby in particular in Examples 17 to 19, 21 to 24,31 and 41, and in WO 02/056905. For the latter application, it is inparticular referred to Example 23, 24, 31 and 51. All three documentsare explicitly incorporated herein by reference.

The present invention also includes HBcAg variants which have beenmodified to delete or substitute one or more additional cysteineresidues. Thus, the vaccine compositions of the invention includecompositions comprising HBcAgs in which cysteine residues not present inthe amino acid sequence shown in SEQ ID NO: 16 have been deleted.

It is well known in the art that free cysteine residues can be involvedin a number of chemical side reactions. These side reactions includedisulfide exchanges, reaction with chemical substances or metabolitesthat are, for example, injected or formed in a combination therapy withother substances, or direct oxidation and reaction with nucleotides uponexposure to UV light. Toxic adducts could thus be generated, especiallyconsidering the fact that HBcAgs have a strong tendency to bind nucleicacids. The toxic adducts would thus be distributed between amultiplicity of species, which individually may each be present at lowconcentration, but reach toxic levels when together.

In view of the above, one advantage to the use of HBcAgs in vaccinecompositions which have been modified to remove naturally residentcysteine residues is that sites to which toxic species can bind whenantigens or antigenic determinants are attached would be reduced innumber or eliminated altogether.

A number of naturally occurring HBcAg variants suitable for use in thepractice of the present invention have been identified. Yuan et al., (J.Virol. 73:10122 10128 (1999)), for example, describe variants in whichthe isoleucine residue at position corresponding to position 97 in SEQID NO:25 is replaced with either a leucine residue or a phenylalanineresidue. The amino acid sequences of a number of HBcAg variants, as wellas several Hepatitis B core antigen precursor variants, are disclosed inGenBank reports AAF121240, AF121239, X85297, X02496, X85305, X85303,AF151735, X85259, X85286, X85260, X85317, X85298, AF043593, M20706,X85295, X80925, X85284, X85275, X72702, X85291, X65258, X85302, M32138,X85293, X85315, U95551, X85256, X85316, X85296, AB033559, X59795,X85299, X85307, X65257, X85311, X85301 (SEQ ID NO:26), X85314, X85287,X85272, X85319, AB010289, X85285, AB010289, AF121242, M90520 (SEQ IDNO:27), P03153, AF110999, and M95589, the disclosures of each of whichare incorporated herein by reference. The sequences of the hereinabovementioned Hepatitis B core antigen precursor variants are furtherdisclosed in WO 01/85208 in SEQ ID NOs: 89-138 of the application WO01/85208. These HBcAg variants differ in amino acid sequence at a numberof positions, including amino acid residues which corresponds to theamino acid residues located at positions 12, 13, 21, 22, 24, 29, 32, 33,35, 38, 40, 42, 44, 45, 49, 51, 57, 58, 59, 64, 66, 67, 69, 74, 77, 80,81, 87, 92, 93, 97, 98, 100, 103, 105, 106, 109, 113, 116, 121, 126,130, 133, 135, 141, 147, 149, 157, 176, 178, 182 and 183 in SEQ ID NO:28. Further HBcAg variants suitable for use in the compositions of theinvention, and which may be further modified according to the disclosureof this specification are described in WO 01/98333, WO 00/177158 and WO00/214478.

HBcAgs suitable for use in the present invention can be derived from anyorganism so long as they are able to enclose or to be coupled orotherwise attached to, in particular as long as they are capable ofpackaging, an unmethylated CpG-containing oligonucleotide and induce animmune response.

As noted above, generally processed HBcAgs (i.e., those which lackleader sequences) will be used in the vaccine compositions of theinvention. The present invention includes vaccine compositions, as wellas methods for using these compositions, which employ the abovedescribed variant HBcAgs.

Further included within the scope of the invention are additional HBcAgvariants which are capable of associating to form dimeric or multimericstructures. Thus, the invention further includes vaccine compositionscomprising HBcAg polypeptides comprising, or alternatively consistingof, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97% or99% identical to any of the wild-type amino acid sequences, and forms ofthese proteins which have been processed, where appropriate, to removethe N terminal leader sequence.

Whether the amino acid sequence of a polypeptide has an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical toone of the wild-type amino acid sequences, or a subportion thereof, canbe determined conventionally using known computer programs such theBestfit program. When using Bestfit or any other sequence alignmentprogram to determine whether a particular sequence is, for instance, 95%identical to a reference amino acid sequence, the parameters are setsuch that the percentage of identity is calculated over the full lengthof the reference amino acid sequence and that gaps in homology of up to5% of the total number of amino acid residues in the reference sequenceare allowed.

The amino acid sequences of the hereinabove mentioned HBcAg variants andprecursors are relatively similar to each other. Thus, reference to anamino acid residue of a HBcAg variant located at a position whichcorresponds to a particular position in SEQ ID NO:28, refers to theamino acid residue which is present at that position in the amino acidsequence shown in SEQ ID NO:28. The homology between these HBcAgvariants is for the most part high enough among Hepatitis B viruses thatinfect mammals so that one skilled in the art would have littledifficulty reviewing both the amino acid sequence shown in SEQ ID NO:28and in SEQ ID NO: 16, respectively, and that of a particular HBcAgvariant and identifying “corresponding” amino acid residues.Furthermore, the HBcAg amino acid sequence shown in SEQ ID NO:27, whichshows the amino acid sequence of a HBcAg derived from a virus whichinfect woodchucks, has enough homology to the HBcAg having the aminoacid sequence shown in SEQ ID NO:28 that it is readily apparent that athree amino acid residue insert is present in SEQ ID NO:27 between aminoacid residues 155 and 156 of SEQ ID NO:28.

The invention also includes vaccine compositions which comprise HBcAgvariants of Hepatitis B viruses which infect birds, as wells as vaccinecompositions which comprise fragments of these HBcAg variants. As oneskilled in the art would recognize, one, two, three or more of thecysteine residues naturally present in these polypeptides could beeither substituted with another amino acid residue or deleted prior totheir inclusion in vaccine compositions of the invention.

As discussed above, the elimination of free cysteine residues reducesthe number of sites where toxic components can bind to the HBcAg, andalso eliminates sites where cross linking of lysine and cysteineresidues of the same or of neighboring HBcAg molecules can occur.Therefore, in another embodiment of the present invention, one or morecysteine residues of the Hepatitis B virus capsid protein have beeneither deleted or substituted with another amino acid residue.Expression and purification of an HBcAg-Lys variant has been describedin Example 24 of WO 02/056905 and the construction of a HBcAg devoid offree cysteine residues and containing an inserted lysine residue hasbeen described in Example 31 of WO 02/056905.

In other embodiments, compositions and vaccine compositions,respectively, of the invention will contain HBcAgs from which the Cterminal region (e.g., amino acid residues 145 185 or 150 185 of SEQ IDNO: 28) has been removed. Thus, additional modified HBcAgs suitable foruse in the practice of the present invention include C terminaltruncation mutants. Suitable truncation mutants include HBcAgs where 1,5, 10, 15, 20, 25, 30, 34, 35, amino acids have been removed from the Cterminus.

HBcAgs suitable for use in the practice of the present invention alsoinclude N terminal truncation mutants. Suitable truncation mutantsinclude modified HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 aminoacids have been removed from the N terminus.

Further HBcAgs suitable for use in the practice of the present inventioninclude N and C terminal truncation mutants. Suitable truncation mutantsinclude HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, and 17 amino acidshave been removed from the N terminus and 1, 5, 10, 15, 20, 25, 30, 34,35 amino acids have been removed from the C terminus.

The invention further includes compositions and vaccine compositions,respectively, comprising HBcAg polypeptides comprising, or alternativelyessentially consisting of, or alternatively consisting of, amino acidsequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identicalto the above described truncation mutants.

In certain embodiments of the invention, a lysine residue is introducedinto a HBcAg polypeptide, to mediate the binding of the antigen orantigenic determinant to the VLP of HBcAg. In preferred embodiments,compositions of the invention are prepared using a HBcAg comprising, oralternatively consisting of, amino acids 1-144, or 1-149, 1-185 of SEQID NO:28, which is modified so that the amino acids corresponding topositions 79 and 80 are replaced with a peptide having the amino acidsequence of Gly-Gly-Lys-Gly-Gly (SEQ ID NO: 18) resulting in the HBcAgpolypeptide having the sequence shown in SEQ ID NO:29). Thesecompositions are particularly useful in those embodiments where anantigenic determinant is coupled to a VLP of HBcAg. In further preferredembodiments, the cysteine residues at positions 48 and 107 of SEQ IDNO:28 are mutated to serine. The invention further includes compositionscomprising the corresponding polypeptides having amino acid sequencesshown in any of the hereinabove mentioned Hepatitis B core antigenprecursor variants which also have above noted amino acid alterations.Further included within the scope of the invention are additional HBcAgvariants which are capable of associating to form a capsid or VLP andhave the above noted amino acid alterations. Thus, the invention furtherincludes compositions and vaccine compositions, respectively, comprisingHBcAg polypeptides which comprise, or alternatively consist of, aminoacid sequences which are at least 80%, 85%, 90%, 95%, 97% or 99%identical to any of the wild-type amino acid sequences, and forms ofthese proteins which have been processed, where appropriate, to removethe N terminal leader sequence and modified with above notedalterations.

Compositions or vaccine compositions of the invention may comprisemixtures of different HBcAgs. Thus, these vaccine compositions may becomposed of HBcAgs which differ in amino acid sequence. For example,vaccine compositions could be prepared comprising a “wild type” HBcAgand a modified HBcAg in which one or more amino acid residues have beenaltered (e.g., deleted, inserted or substituted). Further, preferredvaccine compositions of the invention are those which present highlyordered and repetitive antigen arrays.

As previously disclosed, the invention is based on the surprisingfinding that immunostimulatory substances, and herein in particularspecific DNA-oligonucleotides containing CpG motifs, can be packagedinto VLPs. Unexpectedly, the nucleic acids present in VLPs can bereplaced specifically by the specific DNA-oligonucleotides containingCpG motifs. As an example, the specific CpG-VLPs are more immunogenicand elicit more specific effects than their CpG-free counterparts andinduce enhanced B and T cell responses. The immune response againstantigens coupled, fused or attached otherwise to the VLPs is similarlyenhanced as the immune response against the VLP itself. In addition, theT cell responses against both the VLPs and antigens are especiallydirected to the Th1 type. Furthermore, the packaged nucleic acids andCpGs, respectively, are protected from degradation, i.e., they are morestable. Moreover, non-specific activation of cells from the innateimmune system is dramatically reduced.

The innate immune system has the capacity to recognize invariantmolecular pattern shared by microbial pathogens. Recent studies haverevealed that this recognition is a crucial step in inducing effectiveimmune responses. The main mechanism by which microbial products augmentimmune responses is to stimulate APC, expecially dendritic cells toproduce proinflammatory cytokines and to express high levelscostimulatory molecules for T cells. These activated dendritic cellssubsequently initiate primary T cell responses and dictate the type of Tcell-mediated effector function.

Two classes of nucleic acids, namely 1) bacterial DNA that containsimmunostimulatory sequences, in particular unmethylated CpGdinucleotides within specific flanking bases (referred to as CpG motifs)and 2) double-stranded RNA synthesized by various types of virusesrepresent important members of the microbial components that enhanceimmune responses. Synthetic double stranded (ds) RNA such aspolyinosinic-polycytidylic acid (poly I:C) are capable of inducingdendritic cells to produce proinflammatory cytokines and to express highlevels of costimulatory molecules.

A series of studies by Tokunaga and Yamamoto et al. has shown thatbacterial DNA or synthetic oligodeoxynucleotides induce human PBMC andmouse spleen cells to produce type I interferon (IFN) (reviewed inYamamoto et al., Springer Semin Immunopathol. 22:11-19). Poly (I:C) wasoriginally synthesized as a potent inducer of type I IFN but alsoinduces other cytokines such as IL-12.

Preferred ribonucleic acid encompass polyinosinic-polycytidylic aciddouble-stranded RNA (poly I:C). Ribonucleic acids and modificationsthereof as well as methods for their production have been described byLevy, H. B (Methods Enzymol. 1981, 78:242-251), DeClercq, E (MethodsEnzymol. 1981, 78:227-236) and Torrence, P. F. (Methods Enzymol 1981;78:326-331) and references therein. Further preferred ribonucleic acidscomprise polynucleotides of inosinic acid and cytidiylic acid such poly(IC) of which two strands forms double stranded RNA. Ribonucleic acidscan be isolated from organisms. Ribonucleic acids also encompass furthersynthetic ribonucleic acids, in particular synthetic poly (I:C)oligonucleotides that have been rendered nuclease resistant bymodification of the phosphodiester backbone, in particular byphosphorothioate modifications. In a further embodiment the ribosebackbone of poly (I:C) is replaced by a deoxyribose. Those skilled inthe art know procedures how to synthesize synthetic oligonucleotides.

In another preferred embodiment of the invention molecules that activetoll-like receptors (TLR) are enclosed. Ten human toll-like receptorsare known uptodate. They are activated by a variety of ligands. TLR2 isactivated by peptidoglycans, lipoproteins, lipopolysacchrides,lipoteichonic acid and Zymosan, and macrophage-activating lipopeptideMALP-2; TLR3 is activated by double-stranded RNA such as poly (I:C);TLR4 is activated by lipopolysaccharide, lipoteichoic acids and taxoland heat-shock proteins such as heat shock protein HSP-60 and Gp96; TLR5is activated by bacterial flagella, especially the flagellin protein;TLR6 is activated by peptidoglycans, TLR7 is activated by imiquimoid andimidazoquinoline compounds, such as R-848, loxoribine and bropirimineand TLR9 is activated by bacterial DNA, in particular CpG DNA. Ligandsfor TLR1, TLR8 and TLR10 are not known so far. However, recent reportsindicate that same receptors can react with different ligands and thatfurther receptors are present. The above list of ligands is notexhaustive and further ligands are within the knowledge of the personskilled in the art.

Preferably, the immunostimulatory substance of the present invention isan unmethylated CpG-containing oligonucleotide, wherein the CpG motif ofthe unmethylated CpG-containing oligonucleotide is part of a palindromicsequence, and wherein the palindromic sequence is flanked at its3′-terminus and at its 5′-terminus by less than 10 guanosine entities.In addition, the oligonucleotide preferably comprises about 10 to about30 nucleotides. In a preferred embodiment, the CpG-containingoligonucleotide contains one or more phosphorothioate modifications ofthe phosphate backbone. For example, a CpG-containing oligonucleotidehaving one or more phosphate backbone modifications or having all of thephosphate backbone modified and a CpG-containing oligonucleotide whereinone, some or all of the nucleotide phosphate backbone modifications arephosphorothioate modifications are included within the scope of thepresent invention.

The CpG-containing oligonucleotide can also be recombinant, genomic,synthetic, cDNA, plasmid-derived and single or double stranded. For usein the instant invention, the nucleic acids can be synthesized de novousing any of a number of procedures well known in the art. For example,the b-cyanoethyl phosphoramidite method (Beaucage, S. L., and Caruthers,M. H., Tet. Let. 22:1859 (1981); nucleoside H-phosphonate method (Garegget al., Tet. Let. 27:4051-4054 (1986); Froehler et al., Nucl. Acid. Res.14:5399-5407 (1986); Garegg et al., Tet. Let. 27:4055-4058 (1986),Gaffney et al., Tet. Let. 29:2619-2622 (1988)). These chemistries can beperformed by a variety of automated oligonucleotide synthesizersavailable in the market. Alternatively, CpGs can be produced on a largescale in plasmids, (see Sambrook, T., et al., “Molecular Cloning: ALaboratory Manual,” Cold Spring Harbor laboratory Press, New York, 1989)which after being administered to a subject are degraded intooligonucleotides. Oligonucleotides can be prepared from existing nucleicacid sequences (e.g., genomic or cDNA) using known techniques, such asthose employing restriction enzymes, exonucleases or endonucleases.

The unmethylated CpG-containing oligonucleotide of the invention can bebound to the VLP by any way known in the art provided the compositionenhances an immune response in an animal. For example, theoligonucleotide can be bound either covalently or non-covalently. Inaddition, the VLP can enclose, fully or partially, the unmethylatedCpG-containing oligonucleotide. Preferably, the unmethylatedCpG-containing oligonucleotide can be bound to a VLP site such as anoligonucleotide binding site (either naturally or non-naturallyoccurring), a DNA binding site or a RNA binding site. In anotherembodiment, the VLP site comprises an arginine-rich repeat or alysine-rich repeat.

One specific use for the compositions of the invention is to activatedendritic cells for the purpose of enhancing a specific immune responseagainst antigens. The immune response can be enhanced using ex vivo orin vivo techniques. The ex vivo procedure can be used on autologous orheterologous cells, but is preferably used on autologous cells. Inpreferred embodiments, the dendritic cells are isolated from peripheralblood or bone marrow, but can be isolated from any source of dendriticcells. Ex vivo manipulation of dendritic cells for the purposes ofcancer immunotherapy have been described in several references in theart, including Engleman, E. G., Cytotechnology 25:1 (1997); VanSchooten, W., et al., Molecular Medicine Today, June, 255 (1997);Steinman, R. M., Experimental Hematology 24:849 (1996); and Gluckman, J.C., Cytokines, Cellular and Molecular Therapy 3:187 (1997).

The dendritic cells can also be contacted with the inventivecompositions using in vivo methods. In order to accomplish this, theCpGs are administered in combination with the VLP optionally coupled,fused or otherwise attached to an antigen directly to a subject in needof immunotherapy. In some embodiments, it is preferred that theVLPs/CpGs be administered in the local region of the tumor, which can beaccomplished in any way known in the art, e.g., direct injection intothe tumor.

A further aspect of the present invention and a preferred embodiment ofthe present invention is to provide a composition, typically andpreferably for enhancing an immune response in an animal, comprising (a)a virus-like particle; (b) an immunostimulatory substance; and (c) atleast one antigen or antigenic determinant; wherein said antigen isbound to said virus-like particle; and wherein said . immunostimulatorysubstance is bound to said virus-like particle, and wherein saidimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said palindromic sequence is GACGATCGTC (SEQ ID NO: 1), andwherein said palindromic sequence is flanked at its 3′-terminus and atits 5′-terminus by less than 10 guanosine entities.

We found that the inventive immunostimulatory substances, i.e. theunmethylated CpG-containing oligonucleotides, wherein the CpG motif ofsaid unmethylated CpG-containing oligonucleotides are part of apalindromic sequence, wherein the palindromic sequence is GACGATCGTC(SEQ ID NO: 1), and wherein the palindromic sequence is flanked at its3′-terminus and at its 5′-terminus by less than 10 guanosine entities,are effective at stimulating immune cells in vitro. Moreover, theseinventive immunostimulatory substances have unexpectedly found to bevery efficiently packaged into VLPs. The packaging ability was herebyenhanced as compared to the corresponding immunostimulatory substancehaving the sequence GACGATCGTC (SEQ ID NO: 1) flanked by 10 guanosineentitites at the 5′ and 3′ terminus. The latter was previously found tobe able to stimulate blood cells in vitro (Kuramoto E. et al., JapaneseJournal Cancer Research 83, 1128-1131 (1992).

In a preferred embodiment of the present invention, the palindromicsequence comprises, or alternatively consist essentially of, oralternatively consists of or is GACGATCGTC (SEQ ID NO: 1), wherein saidpalindromic sequence is flanked at its 5′-terminus by at least 3 and atmost 9 guanosine entities and wherein said palindromic sequence isflanked at its 3′-terminus by at least 6 and at most 9 guanosineentities.

In a further very preferred embodiment of the present invention, theimmunostimulatory substance is an umnethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said unmethylated CpG-containing oligonucleotide has a nucleicacid sequence selected from (a) GGGGACGATCGTCGGGGGG ((SEQ ID NO: 2); andtypically abbreviated herein as G3-6), (b) GGGGGACGATCGTCGGGGGG ((SEQ IDNO: 3); and typically abbreviated herein as G4-6), (c)GGGGGGACGATCGTCGGGGGG ((SEQ ID NO: 4); and typically abbreviated hereinas G5-6), (d) GGGGGGGACGATCGTCGGGGGG ((SEQ ID NO: 5); and typicallyabbreviated herein as G6-6), (e) GGGGGGGGACGATCGTCGGGGGGG ((SEQ ID NO:6); and typically abbreviated herein as G7-7), (f)GGGGGGGGGACGATCGTCGGGGGGGG ((SEQ ID NO: 7); and typically abbreviatedherein as G8-8), (g) GGGGGGGGGGACGATCGTCGGGGGGGGG ((SEQ ID NO: 8); andtypically abbreviated herein as G9-9), and (h)GGGGGGCGACGACGATCGTCGTCGGGGGGG ((SEQ ID NO: 9); and typicallyabbreviated herein as G6).

In a further preferred embodiment of the present invention theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said palindromic sequence is GACGATCGTC (SEQ ID NO: 1), andwherein said palindromic sequence is flanked at its 5′-terminus by atleast 4 and at most 9 guanosine entities and wherein said palindromicsequence is flanked at its 3′-terminus by at least 6 and at most 9guanosine entities.

In another preferred embodiment of the present invention theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said unmethylated CpG-containing oligonucleotide has a nucleicacid sequence selected from (a) GGGGGACGATCGTCGGGGGG ((SEQ ID NO: 3);and typically abbreviated herein as G4-6), (b) GGGGGGACGATCGTCGGGGGG((SEQ ID NO: 4); and typically abbreviated herein as G5-6), (c)GGGGGGGACGATCGTCGGGGGG ((SEQ ID NO: 5); and typically abbreviated hereinas G6-6), (d) GGGGGGGGACGATCGTCGGGGGGG ((SEQ ID NO: 6); and typicallyabbreviated herein as G7-7), (e) GGGGGGGGGACGATCGTCGGGGGGG ((SEQ ID NO:7); and typically abbreviated herein as G8-8), (f)GGGGGGGGGGACGATCGTCGGGGGGGGG ((SEQ ID NO: 8); and typically abbreviatedherein as G9-9).

In a further preferred embodiment of the present invention theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said palindromic sequence is GACGATCGTC (SEQ ID NO: 1), andwherein said palindromic sequence is flanked at its 5′-terminus by atleast 5 and at most 8 guanosine entities and wherein said palindromicsequence is flanked at its 3′-terminus by at least 6 and at most 8guanosine entities.

The experimental data show that the ease of packaging of the preferredinventive immunostimulatory substances, i.e. the guanosine flanked,palindromic and unmethylated CpG-containing oligonucleotides, whereinthe palindromic sequence is GACGATCGTC (SEQ ID NO: 1), and wherein thepalindromic sequence is flanked at its 3′-terminus and at its 5′-terminus by less than 10 guanosine entities, into VLP's increases ifthe palindromic sequences are flanked by fewer guanosine entities.However, decreasing the number of guanosine entities flanking thepalindromic sequences leads to a decrease of stimulating blood cells invitro. Thus, packagability is paid by decreased biological activity ofthe indicated inventive immunostimulatory substances. The presentpreferred embodiments represent, thus, a compromise betweenpackagability and biological activity.

In another preferred embodiment of the present invention theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said unmethylated CpG-containing oligonucleotide has a nucleicacid sequence selected from (a) GGGGGGACGATCGTCGGGGGG ((SEQ ID NO: 4);and typically abbreviated herein as G5-6), (b) GGGGGGGACGATCGTCGGGGGG((SEQ ID NO: 5); and typically abbreviated herein as G6-6), (c)GGGGGGGGGACGATCGTCGGGGGGG ((SEQ ID NO: 6); and typically abbreviatedherein as 07-7), (d) GGGGGGGGGACGATCGTCGGGGGGGG ((SEQ ID NO: 7); andtypically abbreviated herein as G8-8).

In a very preferred embodiment of the present invention theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said unmethylated has the nucleic acid sequence of SEQ ID NO: 7,i.e. the immunostimulatory substance is G8-8.

As mentioned above, the optimal sequence used to package into VLPs is acompromise between packagability and biological activity. Taking thisinto consideration, the G8-8 immunostimulatoy substance is a verypreferred embodiment of the present invention since it is biologicallyhighly active while it still reasonably well packaged.

The inventive composition can further comprise an antigen or antigenicdeterminant bound to the virus-like particle. The invention provides forcompositions that vary according to the antigen or antigenic determinantselected in consideration of the desired therapeutic effect. Verypreferred antigens or antigenic determinants suitable for use in thepresent invention are disclosed in WO 00/32227, in WO 01/85208 and in WO02/056905, the disclosures of which are herewith incorporated byreference in their entireties.

The antigen can be any antigen of known or yet unknown provenance. Itcan be isolated from bacteria, viruses or other pathogens or can be arecombinant antigen obtained from expression of suitable nucleic acidcoding therefor. It can also be isolated from prions, tumors,self-molecules, non-peptidic hapten molecules, allergens and hormones.In a preferred embodiment, the antigen is a recombinant antigen. Theselection of the antigen is, of course, dependent upon the immunologicalresponse desired and the host.

In one embodiment of the immune enhancing composition of the presentinvention, the immune response is induced against the VLP itself. Inanother embodiment of the invention a virus-like particle is coupled,fused or otherwise attached to an antigen/immunogen against which anenhanced immune response is desired.

In a further preferred embodiment of the invention, the at least oneantigen or antigenic determinant is fused to the virus-like particle. Asoutlined above, a VLP is typically composed of at least one subunitassembling into a VLP. Thus, in again a further preferred embodiment ofthe invention, the antigen or antigenic determinant is fused to at leastone subunit of the virus-like particle or of a protein capable of beingincorporated into a VLP generating a chimeric VLP-subunit-antigenfusion.

Fusion of the antigen or antigenic determinant can be effected byinsertion into the VLP subunit sequence, or by fusion to either the N-or C-terminus of the VLP-subunit or protein capable of beingincorporated into a VLP. Hereinafter, when referring to fusion proteinsof a peptide to a VLP subunit, the fusion to either ends of the, subunitsequence or internal insertion of the peptide within the subunitsequence are encompassed.

Fusion may also be effected by inserting antigen or antigenicdeterminant sequences into a variant of a VLP subunit where part of thesubunit sequence has been deleted, that are further referred to astruncation mutants. Truncation mutants may have N- or C-terminal, orinternal deletions of part of the sequence of the VLP subunit. Forexample, the specific VLP HBcAg with, for example, deletion of aminoacid residues 79 to 81 is a truncation mutant with an internal deletion.Fusion of antigens or antigenic determinants to either the N- orC-terminus of the truncation mutants VLP-subunits also lead toembodiments of the invention. Likewise, fusion of an epitope into thesequence of the VLP subunit may also be effected by substitution, wherefor example for the specific VLP HBcAg, amino acids 79-81 are replacedwith a foreign epitope. Thus, fusion, as referred to hereinafter, may beeffected by insertion of the antigen or antigenic determinant sequencein the sequence of a VLP subunit, by substitution of part of thesequence of the VLP subunit with the antigen or antigenic determinant,or by a combination of deletion, substitution or insertions.

The chimeric antigen or antigenic determinant -VLP subunit will be ingeneral capable of self-assembly into a VLP. VLP displaying epitopesfused to their subunits are also herein referred to as chimeric VLPs. Asindicated, the virus-like particle comprises or alternatively iscomposed of at least one VLP subunit. In a further embodiment of theinvention, the virus-like particle comprises or alternatively iscomposed of a mixture of chimeric VLP subunits and non-chimeric VLPsubunits, i.e. VLP subunits not having an antigen fused thereto, leadingto so called mosaic particles. This may be advantageous to ensureformation of, and assembly to a VLP. In those embodiments, theproportion of chimeric VLP-subunits may be 1, 2, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 95% or higher.

Flanking amino acid residues may be added to either end of the sequenceof the peptide or epitope to be fused to either end of the sequence ofthe subunit of a VLP, or for internal insertion of such peptidicsequence into the sequence of the subunit of a VLP. Glycine and serineresidues are particularly favored amino acids to be used in the flankingsequences added to the peptide to be fused. Glycine residues conferadditional flexibility, which may diminish the potentially destabilizingeffect of fusing a foreign sequence into the sequence of a VLP subunit.

In a specific embodiment of the invention, the VLP is a Hepatitis B coreantigen VLP. Fusion proteins of the antigen or antigenic determinant toeither the N-terminus of a HBcAg (Neyrinck, S. et al., Nature Med.5:1157-1163 (1999)) or insertions in the so called major immunodominantregion (MIR) have been described (Pumpens, P. and Grens, E.,Intervirology 44:98-114 (2001)), WO 01/98333), and are preferredembodiments of the invention. Naturally occurring variants of HBcAg withdeletions in the MIR have also been described (Pumpens, P. and Grens,E., Intervirology 44:98-114 (2001), which is expressly incorporated byreference in its entirety), and fusions to the N- or C-terminus, as wellas insertions at the position of the MIR corresponding to the site ofdeletion as compared to a wt HBcAg are further embodiments of theinvention. Fusions to the C-terminus have also been described (Pumpens,P. and Grens, E., Intervirology 44:98-114 (2001)). One skilled in theart will easily find guidance on how to construct fusion proteins usingclassical molecular biology techniques (Sambrook, J. et al., eds.,Molecular Cloning, A Laboratory Manual, 2nd. edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), Ho et al., Gene 77:51(1989)). Vectors and plasmids encoding HBcAg and HBcAg fusion proteinsand useful for the expression of a HBcAg and HBcAg fusion proteins havebeen described (Pumpens, P. & Grens, E. Intervirology 44: 98-114 (2001),Neyrinck, S. et al., Nature Med. 5:1157-1163 (1999)) and can be used inthe practice of the invention. An important factor for the optimizationof the efficiency of self-assembly and of the display of the epitope tobe inserted in the MIR of HBcAg is the choice of the insertion site, aswell as the number of amino acids to be deleted from the HBcAg sequencewithin the MLR (Pumpens, P. and Grens, E., Intervirology 44:98-114(2001); EP 421 635; U.S. Pat. No. 6,231,864) upon insertion, or in otherwords, which amino acids form HBcAg are to be substituted with the newepitope. For example, substitution of HBcAg amino acids 76-80, 79-81,79-80, 75-85 or 80-81 with foreign epitopes has been described (Pumpens,P. and Grens, E., Intervirology 44:98-114 (2001); EP 421 635; U.S. Pat.No. 6,231,864). HBcAg contains a long arginine tail (Pumpens, P. andGrens, E., Intervirology 44:98-114 (2001)) which is dispensable forcapsid assembly and capable of binding nucleic acids (Pumpens, P. andGrens, E., Intervirology 44:98-114 (2001)). HBcAg either comprising orlacking this arginine tail are both embodiments of the invention.

In a further preferred embodiment of the invention, the VLP is a VLP ofa RNA phage. The major coat proteins of RNA phages spontaneouslyassemble into VLPs upon expression in bacteria, and in particular in E.coli. Specific examples of bacteriophage coat proteins which can be usedto prepare compositions of the invention include the coat proteins ofRNA bacteriophages such as bacteriophage Qβ (SEQ ID NO:10; PIR Database,Accession No. VCBPQβ referring to Qβ CP and SEQ ID NO: 11; Accession No.AAA16663 referring to Qβ A1 protein) and bacteriophage fr (SEQ ID NO:13; PIR Accession No. VCBPFR). In a more preferred embodiment, the atleast one antigen or antigenic determinant is fused to a Qβ coatprotein. Fusion protein constructs wherein epitopes have been fused tothe C-terminus of a truncated form of the A1 protein of Qβ, or insertedwithin the A1 protein have been described (Kozlovska, T. M., et al.,Intervirology, 39:9-15 (1996)). The A1 protein is generated bysuppression at the UGA stop codon and has a length of 329 aa, or 328 aa,if the cleavage of the N-terminal methionine is taken into account.Cleavage of the N-terminal methionine before an alanine (the secondamino acid encoded by the Qβ CP gene) usually takes place in E. coli,and such is the case for N-termini of the Qβ coat proteins. The part ofthe A1 gene, 3′ of the UGA amber codon encodes the CP extension, whichhas a length of 195 amino acids. Insertion of the at least one antigenor antigenic determinant between position 72 and 73 of the CP extensionleads to further embodiments of the invention (Kozlovska, T. M., et al.,Intervirology 39:9-15 (1996)). Fusion of an antigen or antigenicdeterminant at the C-terminus of a C-terminally truncated Qβ A1 proteinleads to further preferred embodiments of the invention. For example,Kozlovska et al., (Intervirology, 39: 9-15 (1996)) describe Qβ A1protein fusions where the epitope is fused at the C-terminus of the QβCP extension truncated at position 19.

As described by Kozlovska et al. (Intervirology, 39: 9-15 (1996)),assembly of the particles displaying the fused epitopes typicallyrequires the presence of both the A1 protein-antigen fusion and the wtCP to form a mosaic particle. However, embodiments comprising virus-likeparticles, and hereby in particular the VLPs of the RNA phage Qβ coatprotein, which are exclusively composed of VLP subunits having at leastone antigen or antigenic determinant fused thereto, are also within thescope of the present invention.

The production of mosaic particles may be effected in a number of ways.Kozlovska et al., Intervirology, 39:9-15 (1996), describe three methods,which all can be used in the practice of the invention. In the firstapproach, efficient display of the fused epitope on the VLPs is mediatedby the expression of the plasmid encoding the Qβ A1 protein fusionhaving a UGA stop codong between CP and CP extension in a E. coli strainharboring a plasmid encoding a cloned UGA suppressor tRNA which leads totranslation of the UGA codon into Trp (pISM3001 plasmid (Smiley B. K.,et al., Gene 134:33-40 (1993))). In another approach, the CP gene stopcodon is modified into UAA, and a second plasmid expressing the A1protein-antigen fusion is cotransformed. The second plasmid encodes, adifferent antibiotic resistance and the origin of replication iscompatible with the first plasmid (Kozlovska, T. M., et al.,Intervirology 39:9-15 (1996)). In a third approach, CP and the A1protein-antigen fusion are encoded in a bicistronic manner, operativelylinked to a promoter such as the Trp promoter, as described in FIG. 1 ofKozlovska et al., Intervirology, 39:9-15 (1996).

In a further embodiment, the antigen or antigenic determinant isinserted between amino acid 2 and 3 (numbering of the cleaved CP, thatis wherein the N-terminal methionine is cleaved) of the fr CP, thusleading to an antigen or antigenic determinant -fr CP fusion protein.Vectors and expression systems for construction and expression of fr CPfusion proteins self-assembling to VLP and useful in the practice of theinvention have been described (Pushko P. et al., Prot. Eng. 6:883-891(1993)). In a specific embodiment, the antigen or antigenic determinantsequence is inserted into a deletion variant of the fr CP after aminoacid 2, wherein residues 3 and 4 of the fr CP have been deleted (PushkoP. et al., Prot. Eng. 6:883-891 (1993)).

Fusion of epitopes in the N-terminal protuberant β-hairpin of the coatprotein of RNA phage MS-2 and subsequent presentation of the fusedepitope on the self-assembled VLP of RNA phage MS-2 has also beendescribed (WO 92/13081), and fusion of an antigen or antigenicdeterminant by insertion or substitution into the coat protein of MS-2RNA phage is also falling under the scope of the invention.

In another embodiment of the invention, the antigen or antigenicdeterminant is fused to a capsid protein of papillomavirus. In a morespecific embodiment, the antigen or antigenic determinant is fused tothe major capsid protein L1 of bovine papillomavirus type 1 (BPV-1).Vectors and expression systems for construction and expression of BPV-1fusion proteins in a baculovirus/insect cells systems have beendescribed (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA96:2373-2378 (1999), WO 00/23955). Substitution of amino acids 130-136of BPV-1 L1 with an antigen or antigenic determinant leads to a BPV-1L1-antigen fusion protein, which is a preferred embodiment of theinvention. Cloning in a baculovirus vector and expression in baculovirusinfected Sf9 cells has been described, and can be used in the practiceof the invention (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA96:2373-2378 (1999), WO 00/23955). Purification of the assembledparticles displaying the fused antigen or antigenic determinant can beperformed in a number of ways, such as for example gel filtration orsucrose gradient ultracentrifugation (Chackerian, B. et al., Proc. Natl.Acad. Sci. USA 96:2373-2378 (1999), WO 00/23955).

In a further embodiment of the invention, the antigen or antigenicdeterminant is fused to a Ty protein capable of being incorporated intoa Ty VLP. In a more specific embodiment, the antigen or antigenicdeterminant is fused to the p1 or capsid protein encoded by the TYA gene(Roth, J. F., Yeast 16:785-795 (2000)). The yeast retrotransposons Ty1,2, 3 and 4 have been isolated from Saccharomyces Serevisiae, while theretrotransposon Tf1 has been isolated from Schizosaccharomyces Pombae(Boeke, J. D. and Sandmeyer, S. B., “Yeast Transposable elements,” inThe molecular and Cellular Biology of the Yeast Saccharomyces: Genomedynamics, Protein Synthesis, and Energetics, p. 193, Cold Spring HarborLaboratory Press (1991)). The retrotransposons Ty1 and 2 are related tothe copia class of plant and animal elements, while Ty3 belongs to thegypsy family of retrotransposons, which is related to plants and animalretroviruses. In the Ty1 retrotransposon, the p1 protein, also referredto as Gag or capsid protein, has a length of 440 amino acids. P1 iscleaved during maturation of the VLP at position 408, leading to the p2protein, the essential component of the VLP.

Fusion proteins to p1 and vectors for the expression of said fusionproteins in Yeast have been described (Adams, S. E., et al., Nature329:68-70 (1987)). So, for example, an antigen or antigenic determinantmay be fused to p1 by inserting a sequence coding for the antigen orantigenic determinant into the BamH1 site of the pMA5620 plasmid (Adams,S. E., et al., Nature 329:68-70 (1987)). The cloning of sequences codingfor foreign epitopes into the pMA5620 vector leads to expression offusion proteins comprising amino acids 1-381 of p1 of Ty1-15, fusedC-terminally to the N-terminus of the foreign epitope. Likewise,N-terminal fusion of an antigen or antigenic determinant, or internalinsertion into the p1 sequence, or substitution of part of the p1sequence are also meant to fall within the scope of the invention. Inparticular, insertion of an antigen or antigenic determinant into the Tysequence between amino acids 30-31, 67-68, 113-114 and 132-133 of the Typrotein p1 (EP0677111) leads to preferred embodiments of the invention.

Further VLPs suitable for fusion of antigens or antigenic determinantsare, for example, Retrovirus-like-particles (WO9630523), HIV2 Gag (Kang,Y. C., et al, Biol. Chem. 380:353-364 (1999)), Cowpea Mosaic Virus(Taylor, K. M. et al., Biol. Chem. 380:387-392 (1999)), parvovirus VP2VLP (Rueda, P. et al., Virology 263:89-99 (1999)), HBsAg (U.S. Pat. No.4,722,840, EP0201416B1).

Examples of chimeric VLPs suitable for the practice of the invention arealso those described in Intervirology 39:1 (1996). Further examples ofVLPs contemplated for use in the invention are: HPV-1, HPV-6, HPV-11,HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV, HIV GAG, Tobacco MosaicVirus. Virus-like particles of SV-40, Polyomavirus, Adenovirus, HerpesSimplex Virus, Rotavirus and Norwalk virus have also been made, andchimeric VLPs of those VLPs comprising an antigen or antigenicdeterminant are also within the scope of the present invention.

As indicated, embodiments comprising antigens fused to the virus-likeparticle by insertion within the sequence of the virus-like particlebuilding monomer are also within the scope of the present invention. Insome cases, antigens can be inserted in a form of the virus-likeparticle building monomer containing deletions. In these cases, thevirus-like particle building monomer may not be able to form virus-likestructures in the absence of the inserted antigen.

In some instances, recombinant DNA technology can be utilized to fuse aheterologous protein to a VLP protein (Kratz, P. A., et al., Proc. Natl.Acad. Sci. USA 96:1915 (1999)). For example, the present inventionencompasses VLPs recombinantly fused or chemically conjugated (includingboth covalently and non covalently conjugations) to an antigen (orportion thereof, preferably at least 10, 20 or 50 amino acids) of thepresent invention to generate fusion proteins or conjugates. The fusiondoes not necessarily need to be direct, but can occur through linkersequences. More generally, in the case that epitopes, either fused,conjugated or otherwise attached to the virus-like particle, are used asantigens in accordance with the invention, spacer or linker sequencesare typically added at one or both ends of the epitopes. Such linkersequences preferably comprise sequences recognized by the proteasome,proteases of the endosomes or other vesicular compartment of the cell.

One way of coupling is by a peptide bond, in which the conjugate can bea contiguous polypeptide, i.e. a fusion protein. In a fusion proteinaccording to the present invention, different peptides or polypeptidesare linked in frame to each other to form a contiguous polypeptide. Thusa first portion of the fusion protein comprises an antigen or immunogenand a second portion of the fusion protein, either N-terminal orC-terminal to the first portion, comprises a VLP. Alternatively,internal insertion into the VLP, with optional linking sequences on bothends of the antigen, can also be used in accordance with the presentinvention.

When HBcAg is used as the VLP, it is preferred that the antigen islinked to the C-terminal end of the HBcAg particle. The hepatitis B coreantigen (HBcAg) exhibiting a C-terminal fusion of the MHC class Irestricted peptide p33 derived from lymphocytic choriomeningitis virus(LCMV) glycoprotein was used as a model antigen (HBcAg-p33). The 185amino acids long wild type HBc protein assembles into highly structuredparticles composed of 180 subunits assuming icosahedral geometry. Theflexibility of the HBcAg and other VLPs in accepting relatively largeinsertions of foreign sequences at different positions while retainingthe capacity to form structured capsids is well documented in theliterature. This makes the HBc VLPs attractive candidates for the designof non-replicating vaccines.

A flexible linker sequence (e.g. a polyglycine/polyserine-containingsequence such as [Gly4 Ser]2 (Huston et al., Meth. Enzymol 203:46-88(1991)) can be inserted into the fusion protein between the antigen andligand. Also, the fusion protein can be constructed to contain an“epitope tag”, which allows the fusion protein to bind an antibody (e.g.monoclonal antibody) for example for labeling or purification purposes.An example of an epitope tag is a Glu-Glu-Phe tripeptide which isrecognized by the monoclonal antibody YL1/2.

The invention also relates to the chimeric DNA which contains a sequencecoding for the VLP and a sequence coding for the antigen/immunogen. TheDNA can be expressed, for example, in insect cells transformed withBaculoviruses, in yeast or in bacteria. There are no restrictionsregarding the expression system, of which a large selection is availablefor routine use. Preferably, a system is used which allows expression ofthe proteins in large amounts. In general, bacterial expression systemsare preferred on account of their efficiency. One example of a bacterialexpression system suitable for use within the scope of the presentinvention is the one described by Clarke et al., J. Gen. Virol. 71:1109-1117 (1990); Borisova et al., J. Virol. 67: 3696-3701 (1993); andStudier et al., Methods Enzymol. 185:60-89 (1990). An example of asuitable yeast expression system is the one described by Emr, MethodsEnzymol. 185:231-3 (1990); Baculovirus systems, which have previouslybeen used for preparing capsid proteins, are also suitable. Constitutiveor inducible expression systems can be used. By the choice and possiblemodification of available expression systems it is possible to controlthe form in which the proteins are obtained.

In a specific embodiment of the invention, the antigen to which anenhanced immune response is desired is coupled, fused or otherwiseattached in frame to the Hepatitis B virus capsid (core) protein(HBcAg). However, it will be clear to all individuals in the art thatother virus-like particles can be utilized in the fusion proteinconstruct of the invention.

In a further preferred embodiment of the present invention, the at leastone antigen or antigenic determinant is bound to the virus-like particleby at least one covalent bond. Preferably, the least one antigen orantigenic determinant is bound to the virus-like particle by at leastone covalent bond, said covalent bond being a non-peptide bond leadingto an antigen or antigenic determinant array and antigen or antigenicdeterminant -VLP conjugate, respectively. This antigen or antigenicdeterminant array and conjugate, respectively, has typically andpreferably a repetitive and ordered structure since the at least oneantigen or antigenic determinant is bound to the VLP in an orientedmanner. Preferably, equal and more than 120, more preferably equal andmore than 180, even more preferably more than 270, and again morepreferably equal and more than 360 antigens of the invention are boundto the VLP. The formation of a repetitive and ordered antigen orantigenic determinant -VLP array and conjugate, respectively, is ensuredby an oriented and directed as well as defined binding and attachment,respectively, of the at least one antigen or antigenic determinant tothe VLP as will become apparent in the following. Furthermore, thetypical inherent highly repetitive and organized structure of the VLPsadvantageously contributes to the display of the antigen or antigenicdeterminant in a highly ordered and repetitive fashion leading to ahighly organized and repetitive antigen or antigenic determinant -VLParray and conjugate, respectively.

Therefore, the preferred inventive conjugates and arrays, respectively,differ from prior art conjugates in their highly organized structure,dimensions, and in the repetitiveness of the antigen on the surface ofthe array. The preferred embodiment of this invention, furthermore,allows expression of the particle in an expression host guaranteeingproper folding and assembly of the VLP, to which the antigen is thenfurther coupled.

The present invention discloses methods of binding of antigen orantigenic determinant to VLPs. As indicated, in one aspect of theinvention, the at least one antigen or antigenic determinant is bound tothe VLP by way of chemical cross-linking, typically and preferably byusing a heterobifunctional cross-linker. Several hetero bifunctionalcross-linkers are known to the art. In preferred embodiments, thehetero-bifunctional cross-linker contains a functional group which canreact with preferred first attachment sites, i.e. with the side-chainamino group of lysine residues of the VLP or at least one VLP subunit,and a further functional group which can react with a preferred secondattachment site, i.e. a cysteine residue fused to the antigen orantigenic determinant and optionally also made available for reaction byreduction. The first step of the procedure, typically called thederivatization, is the reaction of the VLP with the cross-linker. Theproduct of this reaction is an activated VLP, also called activatedcarrier. In the second step, unreacted cross-linker is removed usingusual methods such as gel filtration or dialysis. In the third step, theantigen or antigenic determinant is reacted with the activated VLP, andthis step is typically called the coupling step. Unreacted antigen orantigenic determinant may be optionally removed in a fourth step, forexample by dialysis. Several hetero-bifunctional cross-linkers are knownto the art. These include the preferred cross-linkers SMPH (Pierce),Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC,SVSB, SIA and other cross-linkers available for example from the PierceChemical Company (Rockford, Ill., USA), and having one functional groupreactive towards amino groups and one functional group reactive towardscysteine residues. The above mentioned cross-linkers all lead toformation of a thioether linkage. Another class of cross-linkerssuitable in the practice of the invention is characterized by theintroduction of a disulfide linkage between the antigen or antigenicdeterminant and the VLP upon coupling. Preferred cross-linkers belongingto this class include for example SPDP and Sulfo-LC-SPDP (Pierce). Theextent of derivatization of the VLP with cross-linker can be influencedby varying experimental conditions such as the concentration of each ofthe reaction partners, the excess of one reagent over the other, the pH,the temperature and the ionic strength. The degree of coupling, i.e. theamount of antigens or antigenic determinants per subunits of the VLP canbe adjusted by varying the experimental conditions described above tomatch the requirements of the vaccine.

A particularly favored method of binding of antigens or antigenicdeterminants to the VLP, is the linking of a lysine residue on thesurface of the VLP with a cysteine residue on the antigen or antigenicdeterminant. In some embodiments, fusion of an amino acid linkercontaining a cysteine residue, as a second attachment site or as a partthereof, to the antigen or antigenic determinant for coupling to the VLPmay be required.

In general, flexible amino acid linkers are favored. Examples of theamino acid linker are selected from the group consisting of: (a) CGG;(b) N-terminal gamma 1-linker; (c) N-terminal gamma 3-linker; (d) Ighinge regions; (e) N-terminal glycine linkers; (f) (G)kC(G)n with n=0-12and k=0-5; (g) N-terminal glycine-serine linkers; (h)(G)kC(G)m(S)l(GGGGS)n with n=0-3, k=0-5, m=0-10, 1=0-2 (SEQ ID NO: 47);(i) GGC; (k) GGC-NH2; (l) C-terminal gamma 1-linker; (m) C-terminalgamma 3-linker; (n) C-terminal glycine linkers; (o) (G)nC(G)k withn=0-12 and k=0-5; (p) C-terminal glycine-serine linkers; (q)(G)m(S)l(GGGGS)n(G)oC(G)k with n=0-3, k=0-5, m=0-10, 1=0-2, and o=0-8(SEQ ID NO: 48).

Further examples of amino acid linkers are the hinge region ofImmunoglobulins, glycine serine linkers (GGGGS)n (SEQ ID NO: 49), andglycine linkers (G)n all further containing a cysteine residue as secondattachment site and optionally further glycine residues. Typicallypreferred examples of said amino acid linkers are N-terminal gamma1:CGDKTHTSPP (SEQ ID NO: 50); C-terminal gamma 1: DKTHTSPPCG (SEQ ID NO:51); N-terminal gamma 3: CGGPKPSTPPGSSGGAP (SEQ ID NO: 52); C-terminalgamma 3: PKPSTPPGSSGGAPGGCG (SEQ ID NO: 53); N-terminal glycine linker:GCGGGG (SEQ ID NO: 54); C-terminal glycine linker: GGGGCG (SEQ ID NO:55); C-terminal glycine-lysine linker: GGKKGC (SEQ ID NO: 56);N-terminal glycine-lysine linker: CGKKGG (SEQ ID NO: 57).

Other amino acid linkers particularly suitable in the practice of theinvention, when a hydrophobic antigen or antigenic determinant is boundto a VLP, are CGKKGG (SEQ ID NO: 58), or CGDEGG (SEQ ID NO: 59) forN-terminal linkers, or GGKKGC (SEQ ID NO: 60) and GGEDGC (SEQ ID NO:61), for the C-terminal linkers. For the C-terminal linkers, theterminal cysteine is optionally C-terminally amidated.

In preferred embodiments of the present invention, GGCG (SEQ ID NO: 62),GGC or GGC-NH2 (“NH2” stands for amidation) linkers at the C-terminus ofthe peptide or CGG at its N-terminus are preferred as amino acidlinkers. In general, glycine residues will be inserted between bulkyamino acids and the cysteine to be used as second attachment site, toavoid potential steric hindrance of the builder amino acid in thecoupling reaction. In the most preferred embodiment of the invention,the amino acid linker GGC-NH2 is fused to the C-terminus of the antigenor antigenic determinant.

The cysteine residue present on the antigen or antigenic determinant hasto be in its reduced state to react with the hetero-bifunctionalcross-linker on the activated VLP, that is a free cysteine or a cysteineresidue with a free sulfhydryl group has to be available. In theinstance where the cysteine residue to function as binding site is in anoxidized form, for example if it is forming a disulfide bridge,reduction of this disulfide bridge with e.g. DTT, TCEP orβ-mercaptoethanol is required. Low concentrations of reducing agent arecompatible with coupling as described in WO 02/05690, higherconcentrations inhibit the coupling reaction, as a skilled artisan wouldknow, in which case the reductand has to be removed or its concentrationdecreased prior to coupling, e.g. by dialysis, gel filtration or reversephase HPLC.

Binding of the antigen or antigenic determinant to the VLP by using ahetero-bifunctional cross-linker according to the preferred methodsdescribed above, allows coupling of the antigen or antigenic determinantto the VLP in an oriented fashion. Other methods of binding the antigenor antigenic determinant to the VLP include methods wherein the antigenor antigenic determinant is cross-linked to the VLP using thecarbodiimide EDC, and NHS. In further methods, the antigen or antigenicdeterminant is attached to the VLP using a homo-bifunctionalcross-linker such as glutaraldehyde, DSG, BM[PEO]4, BS3, (PierceChemical Company, Rockford, Ill., USA) or other known homo-bifunctionalcross-linkers with functional groups reactive towards amine groups orcarboxyl groups of the VLP.

Other methods of binding the VLP to an antigen or antigenic determinantinclude methods where the VLP is biotinylated, and the antigen orantigenic determinant expressed as a streptavidin-fusion protein, ormethods wherein both the antigen or antigenic determinant and the VLPare biotinylated, for example as described in WO 00/23955. In this case,the antigen or antigenic determinant may be first bound to streptavidinor avidin by adjusting the ratio of antigen or antigenic determinant tostreptavidin such that free binding sites are still available forbinding of the VLP, which is added in the next step. Alternatively, allcomponents may be mixed in a “one pot” reaction. Other ligand-receptorpairs, where a soluble form of the receptor and of the ligand isavailable, and are capable of being cross-linked to the VLP or theantigen or antigenic determinant, may be used as binding agents forbinding antigen or antigenic determinant to the VLP. Alternatively,either the ligand or the receptor may be fused to the antigen orantigenic determinant, and so mediate binding to the VLP chemicallybound or fused either to the receptor, or the ligand respectively.Fusion may also be effected by insertion or substitution.

As already indicated, in a favored embodiment of the present invention,the VLP is the VLP of a RNA phage, and in a more preferred embodiment,the VLP is the VLP of RNA phage Qβ coat protein.

One or several antigen molecules, i.e. one or several antigens orantigenic determinants, can be attached to one subunit of the capsid orVLP of RNA phages coat proteins, preferably through the exposed lysineresidues of the VLP of RNA phages, if sterically allowable. A specificfeature of the VLP of the coat protein of RNA phages and in particularof the Qβ coat protein VLP is thus the possibility to couple severalantigens per subunit. This allows for the generation of a dense antigenarray.

In a preferred embodiment of the invention, the binding and attachment,respectively, of the at least one antigen or antigenic determinant tothe virus-like particle is by way of interaction and association,respectively, between at least one first attachment site of thevirus-like particle and at least one second attachment of the antigen orantigenic determinant.

VLPs or capsids of Qβ coat protein display a defined number of lysineresidues on their surface, with a defined topology with three lysineresidues pointing towards the interior of the capsid and interactingwith the RNA, and four other lysine residues exposed to the exterior ofthe capsid. These defined properties favor the attachment of antigens tothe exterior of the particle, rather than to the interior of theparticle where the lysine residues interact with RNA. VLPs of other RNAphage coat proteins also have a defined number of lysine residues ontheir surface and a defined topology of these lysine residues.

In further preferred embodiments of the present invention, the firstattachment site is a lysine residue and/or the second attachmentcomprises sulfhydryl group or a cysteine residue; In a very preferredembodiment of the present invention, the first attachment site is alysine residue and the second attachment is a cysteine residue.

In very preferred embodiments of the invention, the antigen or antigenicdeterminant is bound via a cysteine residue, to lysine residues of theVLP of RNA phage coat protein, and in particular to the VLP of Qβ coatprotein.

Another advantage of the VLPs derived from RNA phages is their highexpression yield in bacteria that allows production of large quantitiesof material at affordable cost.

As indicated, the inventive conjugates and arrays, respectively, differfrom prior art conjugates in their highly organized structure,dimensions, and in the repetitiveness of the antigen on the surface ofthe array. Moreover, the use of the VLPs as carriers allow the formationof robust antigen arrays and conjugates, respectively, with variableantigen density. In particular, the use of VLPs of RNA phages, andhereby in particular the use of the VLP of RNA phage Qβ coat proteinallows to achieve very high epitope density. In particular, a density ofmore than 1.5 epitopes per subunit has been reached by coupling forexample the human Aβ1-6 peptide to the VLP of Qβ coat protein (WO2004/016282). The preparation of compositions of VLPs of RNA phage coatproteins with a high epitope density can be effected using the teachingof this application. In preferred embodiment of the invention, when anantigen or antigenic determinant is coupled to the VLP of Qβ coatprotein, an average number of antigen or antigenic determinant persubunit of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 2.5, 2.6, 2.7, 2.8, 2.9, orhigher is preferred.

The second attachment site, as defined herein, may be either naturallyor non-naturally present with the antigen or the antigenic determinant.In the case of the absence of a suitable natural occurring secondattachment site on the antigen or antigenic determinant, such a, thennon-natural second attachment has to be engineered to the antigen.

As described above, four lysine residues are exposed on the surface ofthe VLP of Qβ coat protein. Typically these residues are derivatizedupon reaction with a cross-linker molecule. In the instance where notall of the exposed lysine residues can be coupled to an antigen, thelysine residues which have reacted with the cross-linker are left with across-linker molecule attached to the □-amino group after thederivatization step. This leads to disappearance of one or severalpositive charges, which may be detrimental to the solubility andstability of the VLP. By replacing some of the lysine residues witharginines, as in the disclosed Qβ coat protein mutants described below,we prevent the excessive disappearance of positive charges since thearginine residues do not react with the cross-linker. Moreover,replacement of lysine residues by arginines may lead to more definedantigen arrays, as fewer sites are available for reaction to theantigen.

Accordingly, exposed lysine residues were replaced by arginines in thefollowing Qβ coat protein mutants and mutant Qβ VLPs disclosed in thisapplication: Qβ-240 (Lys13-Arg; SEQ ID NO:20), Qβ-250 (Lys 2-Arg,Lys13-Arg; SEQ ID NO: 22) and Qβ-259 (Lys 2-Arg, Lys16-Arg; SEQ IDNO:24). The constructs were cloned, the proteins expressed, the VLPspurified and used for coupling to peptide and protein antigens. Qβ-251;(SEQ ID NO: 23) was also constructed, and guidance on how to express,purify and couple the VLP of Qβ-251 coat protein can be found throughoutthe application.

In a further embodiment, we disclose a Qβ mutant coat protein with oneadditional lysine residue, suitable for obtaining even higher densityarrays of antigens. This mutant Qβ coat protein, Qβ-243 (Asn 10-Lys; SEQID NO: 21), was cloned, the protein expressed, and the capsid or VLPisolated and purified, showing that introduction of the additionallysine residue is compatible with self-assembly of the subunits to acapsid or VLP. Thus, antigen or antigenic determinant arrays andconjugates, respectively, may be prepared using VLP of Qβ coat proteinmutants. A particularly favored method of attachment of antigens toVLPs, and in particular to VLPs of RNA phage coat proteins is thelinking of a lysine residue present on the surface of the VLP of RNAphage coat proteins with a cysteine residue added to the antigen. Inorder for a cysteine residue to be effective as second attachment site,a sulfhydryl group must be available for coupling. Thus, a cysteineresidue has to be in its reduced state, that is, a free cysteine or acysteine residue with a free sulfhydryl group has to be available. Inthe instant where the cysteine residue to function as second attachmentsite is in an oxidized form, for example if it is forming a disulfidebridge, reduction of this disulfide bridge with e.g. DTT, TCEP orβ-mercaptoethanol is required. The concentration of reductand, and themolar excess of reductand over antigen has to be adjusted for eachantigen. A titration range, starting from concentrations as low as 10 μMor lower, up to 10 to 20 mM or higher reductand if required is tested,and coupling of the antigen to the carrier assessed. Although lowconcentrations of reductand are compatible with the coupling reaction asdescribed in WO 02/056905, higher concentrations inhibit the couplingreaction, as a skilled artisan would know, in which case the reductandhas to be removed or its concentration decreased, e.g. by dialysis, gelfiltration or reverse phase HPLC. Advantageously, the pH of the dialysisor equilibration buffer is lower than 7, preferably 6. The compatibilityof the low pH buffer with antigen activity or stability has to betested.

Epitope density on the VLP of RNA phage coat proteins can be modulatedby the choice of cross-linker and other reaction conditions. Forexample, the cross-linkers Sulfo-GMBS and SMPH typically allow reachinghigh epitope density. Derivatization is positively influenced by highconcentration of reactands, and manipulation of the reaction conditionscan be used to control the number of antigens coupled to VLPs of RNAphage coat proteins, and in particular to VLPs of Qβ coat protein.

Prior to the design of a non-natural second attachment site the positionat which it should be fused, inserted or generally engineered has to bechosen. The selection of the position of the second attachment site may,by way of example, be based on a crystal structure of the antigen. Sucha crystal structure of the antigen may provide information on theavailability of the C- or N-termini of the molecule (determined forexample from their accessibility to solvent), or on the exposure tosolvent of residues suitable for use as second attachment sites, such ascysteine residues. Exposed disulfide bridges, as is the case for Fabfragments, may also be a source of a second attachment site, since theycan be generally converted to single cysteine residues through mildreduction, with e.g. 2-mercaptoethylamine, TCEP, β-mercaptoethanol orDTT. Mild reduction conditions not affecting the immunogenicity of theantigen will be chosen. In general, in the case where immunization witha self-antigen is aiming at inhibiting the interaction of thisself-antigen with its natural ligands, the second attachment site willbe added such that it allows generation of antibodies against the siteof interaction with the natural ligands. Thus, the location of thesecond attachment site will be selected such that steric hindrance fromthe second attachment site or any amino acid linker containing the sameis avoided. In further embodiments, an antibody response directed at asite distinct from the interaction site of the self-antigen with itsnatural ligand is desired. In such embodiments, the second attachmentsite may be selected such that it prevents generation of antibodiesagainst the interaction site of the self-antigen with its naturalligands.

Other criteria in selecting the position of the second attachment siteinclude the oligomerization state of the antigen, the site ofoligomerization, the presence of a cofactor, and the availability ofexperimental evidence disclosing sites in the antigen structure andsequence where modification of the antigen is compatible with thefunction of the self-antigen, or with the generation of antibodiesrecognizing the self-antigen.

In very preferred embodiments, the antigen or antigenic determinantcomprises a single second attachment site or a single reactiveattachment site capable of association with the first attachment siteson the core particle and the VLPs or VLP subunits, respectively. Thisfurther ensures a defined and uniform binding and association,respectively, of the at least one, but typically more than one,preferably more than 10, 20, 40, 80, 120, 150, 180, 210, 240, 270, 300,360, 400, 450 antigens to the core particle and VLP, respectively. Theprovision of a single second attachment site or a single reactiveattachment site on the antigen, thus, ensures a single and uniform typeof binding and association, respectively leading to a very highlyordered and repetitive array. For example, if the binding andassociation, respectively, is effected by way of a lysine—(as the firstattachment site) and cysteine—(as a second attachment site) interaction,it is ensured, in accordance with this preferred embodiment of theinvention, that only one cysteine residue per antigen, independentwhether this cysteine residue is naturally or non-naturally present onthe antigen, is capable of binding and associating, respectively, withthe VLP and the first attachment site of the core particle,respectively.

In some embodiments, engineering of a second attachment site onto theantigen require the fusion of an amino acid linker containing an aminoacid suitable as second attachment site according to the disclosures ofthis invention. Therefore, in a preferred embodiment of the presentinvention, an amino acid linker is bound to the antigen or the antigenicdeterminant by way of at least one covalent bond. Preferably, the aminoacid linker comprises, or alternatively consists of, the secondattachment site. In a further preferred embodiment, the amino acidlinker comprises a sulfhydryl group or a cysteine residue. In anotherpreferred embodiment, the amino acid linker is cysteine. Some criteriaof selection of the amino acid linker as well as further preferredembodiments of the amino acid linker according to the invention havealready been mentioned above.

In another specific embodiment of the invention, the attachment site isselected to be a lysine or cysteine residue that is fused in frame tothe HBcAg. In a preferred embodiment, the antigen is fused to theC-terminus of HBcAg via a three leucine linker.

When an antigen or antigenic determinant is linked to the VLP through alysine residue, it may be advantageous to either substitute or deleteone or more of the naturally resident lysine residues, as well as otherlysine residues present in HBcAg variants.

In many instances, when the naturally resident lysine residues areeliminated, another lysine will be introduced into the HBcAg as anattachment site for an antigen or antigenic determinant. Methods forinserting such a lysine residue are known in the art. Lysine residuesmay also be added without removing existing lysine residues.

The C terminus of the HBcAg has been shown to direct nuclearlocalization of this protein. (Eckhardt et al., J. Virol. 65:575 582(1991)). Further, this region of the protein is also believed to conferupon the HBcAg the ability to bind nucleic acids.

As indicated, HBcAgs suitable for use in the practice of the presentinvention also include N terminal truncation mutants. Suitabletruncation mutants include modified HBcAgs where 1, 2, 5, 7, 9, 10, 12,14, 15, or 17 amino acids have been removed from the N terminus.However, variants of virus-like particles containing internal deletionswithin the sequence of the subunit composing the virus-like particle arealso suitable in accordance with the present invention, provided theircompatibility with the ordered or particulate structure of thevirus-like particle. For example, internal deletions within the sequenceof the HBcAg are suitable (Preikschat, P., et al., J. Gen. Virol.80:1777-1788 (1999)).

Further HBcAgs suitable for use in the practice of the present inventioninclude N- and C terminal truncation mutants. Suitable truncationmutants include HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, and 17 aminoacids have been removed from the N terminus and 1, 5, 10, 15, 20, 25,30, 34, 35, 36, 37, 38, 39 40, 41, 42 or 48 amino acids have beenremoved from the C terminus.

Vaccine compositions of the invention can comprise mixtures of differentHBcAgs. Thus, these vaccine compositions can be composed of HBcAgs whichdiffer in amino acid sequence. For example, vaccine compositions couldbe prepared comprising a “wild type” HBcAg and a modified HBcAg in whichone or more amino acid residues have been altered (e.g., deleted,inserted or substituted). In most applications, however, only one typeof a HBcAg will be used.

In a preferred embodiment, the virus-like particle comprises at leastone first attachment site and the antigen or antigenic determinantcomprises at least one second attachment site. Preferably, the firstattachment site comprises, or preferably consists of, an amino group ora lysine residue. The second attachment site is preferably selected fromthe group consisting of (a) an attachment site not naturally occurringwith said antigen or antigenic determinant; and (b) an attachment sitenaturally occurring with said antigen or antigenic determinant. Evenmore preferably, the second attachment site comprises, or preferablyconsists of, a sulfhydryl group or a cysteine residue. In a preferredembodiment, the binding of the antigen or antigenic determinant to thevirus-like particle is effected through association between the firstattachment site and the second attachment site, wherein preferably theassociation is through at least one non-peptide bond, and whereinpreferably the antigen or antigenic determinant and the virus-likeparticle interact through said association to form an ordered andrepetitive antigen array. In one embodiment, the first attachment siteis a lysine residue and the second attachment site is a cysteineresidue. In another embodiment, the first attachment site is an aminogroup and the second attachment site is a sulfhydryl group.

In a specific embodiment of the invention, the antigen or antigenicdeterminat, comprises one or more cytotoxic T cell epitopes, Th cellepitopes, or a combination of the two epitopes. Thus, in one embodiment,the antigen or antigenic determinant comprises one, two, or morecytotoxic T cell epitopes. In another embodiment, the antigen orantigenic determinant comprises one, two, or more Th cell epitopes. Inyet another embodiment, the antigen or antigenic determinant comprisesone, two or more cytotoxic T cell epitopes and one, two or more Th cellepitopes.

The present invention is applicable to a wide variety of antigens. In apreferred embodiment, the antigen is a protein, polypeptide or peptide.In another embodiment the antigen is DNA. The antigen can also be alipid, a carbohydrate, or an organic molecule, in particular a smallorganic molecule such as nicotine.

Antigens of the invention can be selected from the group consisting ofthe following: (a) polypeptides suited to induce an immune responseagainst cancer cells; (b) polypeptides suited to induce an immuneresponse against infectious diseases; (c) polypeptides suited to inducean immune response against allergens; (d) polypeptides suited to inducean immune response in farm animals or pets; and (e) fragments (e.g., adomain) of any of the polypeptides set out in (a) (d).

Preferred antigens include those from a pathogen (e.g. virus, bacterium,parasite, fungus) and tumors (especially tumor-associated antigens or“tumor markers”). Other preferred antigens are autoantigens and selfantigens, respectively.

In the specific embodiments described in the Examples, the antigen isthe peptide p33 derived from lymphocytic choriomeningitis virus (LCMV).The p33 peptide represents one of the best studied CTL epitopes (Pircheret al., “Tolerance induction in double specific T-cell receptortransgenic mice varies with antigen,” Nature 342:559 (1989); Tissot etal., “Characterizing the functionality of recombinant T-cell receptorsin vitro: a pMHC tetramer based approach,” J Immunol Methods 236:147(2000); Bachmann et al., “Four types of Ca2+-signals after stimulationof naive T cells with T cell agonists, partial agonists andantagonists,” Eur. J. Immunol. 27:3414 (1997); Bachmann et al.,“Functional maturation of an anti-viral cytotoxic T cell response,” J.Virol. 71:5764 (1997); Bachmann et al., “Peptide induced TCR-downregulation on naive T cell predicts agonist/partial agonist propertiesand strictly correlates with T cell activation,” Eur. J. Immunol.27:2195 (1997); Bachmann et al., “Distinct roles for LFA-1 and CD28during activation of naive T cells: adhesion versus costimulation,”Immunity 7:549 (1997)). p33-specific T cells have been shown to inducelethal diabetic disease in transgenic mice (Ohashi et al., “Ablation of‘tolerance’ and induction of diabetes by virus infection in viralantigen transgenic mice,” Cell 65:305 (1991)) as well as to be able toprevent growth of tumor cells expressing p33 (Kündig et al.,“Fibroblasts act as efficient antigen-presenting cells in lymphoidorgans,” Science 268:1343 (1995); Speiser et al., “CTL tumor therapyspecific for an endogenous antigen does not cause autoimmune disease,”J. Exp. Med. 186:645 (1997)). This specific epitope, therefore, isparticularly well suited to study autoimmunity, tumor immunology as wellas viral diseases.

In one specific embodiment of the invention, the antigen or antigenicdeterminant is one that is useful for the prevention of infectiousdisease. Such treatment will be useful to treat a wide variety ofinfectious diseases affecting a wide range of hosts, e.g., human, cow,sheep, pig, dog, cat, other mammalian species and non-mammalian speciesas well. Treatable infectious diseases are well known to those skilledin the art, and examples include infections of viral etiology such asHIV, influenza, Herpes, viral hepatitis, Epstein Bar, polio, viralencephalitis, measles, chicken pox, Papilloma virus etc.; or infectionsof bacterial etiology such as pneumonia, tuberculosis, syphilis, etc.;or infections of parasitic etiology such as malaria, trypanosomiasis,leishmaniasis, trichomoniasis, amoebiasis, etc. Thus, antigens orantigenic determinants selected for the compositions of the inventionwill be well known to those in the medical art; examples of antigens orantigenic determinants include the following: the HIV antigens gp140 andgp160; the influenza antigens hemagglutinin, M2 protein andneuraminidase, Hepatitis B surface antigen or core and circumsporozoiteprotein of malaria or fragments thereof.

As discussed above, antigens include infectious microbes such asviruses, bacteria and fungi and fragments thereof, derived from naturalsources or synthetically. Infectious viruses of both human and non-humanvertebrates include retroviruses, RNA viruses and DNA viruses. The groupof retroviruses includes both simple retroviruses and complexretroviruses. The simple retroviruses include the subgroups of B-typeretroviruses, C-type retroviruses and D-type retroviruses. An example ofa B-type retrovirus is mouse mammary tumor virus (MMTV). The C-typeretroviruses include subgroups C-type group A (including Rous sarcomavirus (RSV), avian. leukemia virus (ALV), and avian myeloblastosis virus(AMV)) and C-type group B (including murine leukemia virus (MLV), felineleukemia virus (FeLV), murine sarcoma virus (MSV), gibbon ape leukemiavirus (GALV), spleen necrosis virus (SNV), reticuloendotheliosis virus(RV) and simian sarcoma virus (SSV)). The D-type retroviruses include.Mason-Pfizer monkey virus (MPMV) and simian retrovirus type 1 (SRV-1).The complex retroviruses include the subgroups of lentiviruses, T-cellleukemia viruses and the foamy viruses. Lentiviruses include HIV-1, butalso include HIV-2, SIV, Visna virus, feline immunodeficiency virus(PTV), and equine infectious anemia virus (EIAV). The T-cell leukemiaviruses include HTLV-1, HTLV-II, simian T-cell leukemia virus (STLV),and bovine leukemia virus (BLV). The foamy viruses include human foamyvirus (HFV), simian foamy virus (SFV) and bovine foamy virus (BFV).

Examples of RNA viruses that are antigens in vertebrate animals include,but are not limited to, the following: members of the family Reoviridae,including the genus Orthoreovirus (multiple serotypes of both mammalianand avian retroviruses), the genus Orbivirus (Bluetongue virus,Eugenangee virus, Kemerovo virus, African horse sickness virus, andColorado Tick Fever virus), the genus Rotavirus (human rotavirus,Nebraska calf diarrhea virus, murine rotavirus, simian rotavirus, bovineor ovine rotavirus, avian rotavirus); the family Picomaviridae,including the genus Enterovirus (poliovirus, Coxsackie virus A and B,enteric cytopathic human orphan (ECHO) viruses, hepatitis A,

C, D, E and G viruses, Simian enteroviruses, Murine encephalomyelitis(ME) viruses, Poliovirus muris, Bovine enteroviruses, Porcineenteroviruses, the genus Cardiovirus (Encephalomyocarditis virus (EMC),Mengovirus), the genus Rhinovirus (Human rhinoviruses including at least113 subtypes; other rhinoviruses), the genus Apthovirus (Foot and Mouthdisease (FMDV); the family Calciviridae, including Vesicular exanthemaof swine virus, San Miguel sea lion virus, Feline picornavirus andNorwalk virus; the family Togaviridae, including the genus Alphavirus(Eastern equine encephalitis virus, Semliki forest virus, Sindbis virus,Chikungunya virus, O'Nyong-Nyong virus, Ross river virus, Venezuelanequine encephalitis virus, Western equine encephalitis virus), the genusFlavirius (Mosquito borne yellow fever virus, Dengue virus,

Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valleyencephalitis virus, West Nile virus, Kunjin virus, Central European tickborne virus, Far Eastern tick borne virus, Kyasanur forest virus,Louping III virus, Powassan virus, Omsk hemorrhagic fever virus), thegenus Rubivirus (Rubella virus), the genus Pestivirus (Mucosal diseasevirus, Hog cholera virus, Border disease virus); the familyBunyaviridae, including the genus Bunyvirus (Bunyamwera and relatedviruses, California encephalitis group viruses), the genus Phlebovirus(Sandfly fever Sicilian virus, Rift Valley fever virus), the genusNairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep diseasevirus), and the genus Uukuvirus (Uukuniemi and related viruses); thefamily Orthomyxoviridae, including the genus Influenza virus (Influenzavirus type A, many human subtypes); Swine influenza virus, and Avian andEquine Influenza viruses; influenza type B (many human subtypes), andinfluenza type C (possible separate genus); the family paramyxoviridae,including the genus Paramyxovirus (Parainfluenza virus type 1, Sendaivirus, Hemadsorption virus, Parainfluenza viruses types 2 to 5,Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measlesvirus, subacute sclerosing panencephalitis virus, distemper virus,Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus(RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice);forest virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong virus,Ross river virus, Venezuelan equine encephalitis virus, Western equineencephalitis virus), the genus Flavirius (Mosquito borne yellow fevervirus, Dengue virus, Japanese encephalitis virus, St. Louis encephalitisvirus, Murray Valley encephalitis virus, West Nile virus, Kunjin virus,Central European tick borne virus, Far Eastern tick borne virus,Kyasanur forest virus, Louping III virus, Powassan virus, Omskhemorrhagic fever virus), the genus Rubivirus (Rubella virus), the genusPestivirus (Mucosal disease virus, Hog cholera virus, Border diseasevirus); the family Bunyaviridae, including the genus Bunyvirus(Bunyamwera and related viruses, California encephalitis group viruses),the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fevervirus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus,Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi andrelated viruses); the family Orthomyxoviridae, including the genusInfluenza virus (Influenza virus type A, many human subtypes); Swineinfluenza virus, and Avian and Equine Influenza viruses; influenza typeB (many human subtypes), and influenza type C (possible separate genus);the family paramyxoviridae, including the genus Paramyxovirus(Parainfluenza virus type 1, Sendai virus, Hemadsorption virus,Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumpsvirus), the genus Morbillivirus (Measles virus, subacute sclerosingpanencephalitis virus, distemper virus, Rinderpest virus), the genusPneumovirus (respiratory syncytial virus (RSV), Bovine respiratorysyncytial virus and Pneumonia virus of mice); the family Rhabdoviridae,including the genus Vesiculovirus (VSV), Chandipura virus, Flanders-HartPark virus), the genus Lyssavirus (Rabies virus), fish Rhabdoviruses andfiloviruses (Marburg virus and Ebola virus); the family Arenaviridae,including Lymphocytic choriomeningitis virus (LCM), Tacaribe viruscomplex, and Lassa virus; the family Coronoaviridae, includingInfectious Bronchitis Virus (IBV), Mouse Hepatitis virus, Human entericcorona virus, and Feline infectious peritonitis (Feline coronavirus).

Illustrative DNA viruses that are antigens in vertebrate animalsinclude, but are not limited to: the family Poxviridae, including thegenus Orthopoxvirus (Variola major, Variola minor, Monkey pox Vaccinia,Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus(Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avianpoxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genusSuipoxvirus (Swinepox), the genus Parapoxvirus (contagious postulardermatitis virus, pseudocowpox, bovine papular stomatitis virus); thefamily Iridoviridae (African swine fever virus, Frog viruses 2 and 3,Lymphocystis virus of fish); the family Herpesviridae, including thealpha-Herpesviruses (Herpes Simplex Types 1 and 2, Varicella-Zoster,Equine abortion virus, Equine herpes virus 2 and 3, pseudorabies virus,infectious bovine keratoconjunctivitis virus, infectious bovinerhinotracheitis virus, feline rhinotracheitis virus, infectiouslaryngotracheitis virus) the Beta-herpesviruses (Human cytomegalovirusand cytomegaloviruses of swine, monkeys and rodents); thegamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease virus,Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus, guinea pigherpes virus, Lucke tumor virus); the family Adenoviridae, including thegenus Mastadenovirus (Human subgroups A, B, C, D and E and ungrouped;simian adenoviruses (at least 23 serotypes), infectious caninehepatitis, and adenoviruses of cattle, pigs, sheep, frogs and many otherspecies, the genus Aviadenovirus (Avian adenoviruses); andnon-cultivatable adenoviruses; the family Papoviridae, including thegenus Papillomavirus (Human papilloma viruses, bovine papilloma viruses,Shope rabbit papilloma virus, and various pathogenic papilloma virusesof other species), the genus Polyomavirus (polyomavirus, Simianvacuolating agent (SV-40), Rabbit vacuolating agent (RKV), K virus, BKvirus, JC virus, and other primate polyoma viruses such as Lymphotrophicpapilloma virus); the family Parvoviridae including the genusAdeno-associated viruses, the genus Parvovirus (Feline panleukopeniavirus, bovine parvovirus, canine parvovirus, Aleutian mink diseasevirus, etc.). Finally, DNA viruses may include viruses which do not fitinto the above families such as Kuru and Creutzfeldt-Jacob diseaseviruses and chronic infectious neuropathic agents (CHINA virus).

Each of the foregoing lists is illustrative, and is not intended to belimiting. In a specific embodiment of the invention, the antigencomprises one or more cytotoxic T cell epitopes, Th cell epitopes, or acombination of the two epitopes.

In addition to enhancing an antigen specific immune response in humans,the methods of the preferred embodiments are particularly well suitedfor treatment of other mammals or other animals, e.g., birds such ashens, chickens, turkeys, ducks, geese, quail and pheasant. Birds areprime targets for many types of infections.

An example of a common infection in chickens is chicken infectiousanemia virus (CIAV). CIAV was first isolated in Japan in 1979 during aninvestigation of a Marek's disease vaccination break (Yuasa et al.,Avian Dis. 23:366-385 (1979)). Since that time, CIAV has been detectedin commercial poultry in all major poultry producing countries (vanBulow et al., pp. 690-699 in “Diseases of Poultry”, 9th edition, IowaState University Press 1991).

Vaccination of birds, like other vertebrate animals can be performed atany age. Normally, vaccinations are performed at up to 12 weeks of agefor a live microorganism and between 14-18 weeks for an inactivatedmicroorganism or other type of vaccine. For in ovo vaccination,vaccination can be performed in the last quarter of embryo development.The vaccine can be administered subcutaneously, by spray, orally,intraocularly, intratracheally, nasally, in ovo or by other methodsdescribed herein.

Cattle and livestock are also susceptible to infection. Disease whichaffect these animals can produce severe economic losses, especiallyamongst cattle. The methods of the invention can be used to protectagainst infection in livestock, such as cows, horses, pigs, sheep andgoats.

Cows can be infected by bovine viruses. Bovine viral diarrhea virus(BVDV) is a small enveloped positive-stranded RNA virus and isclassified, along with hog cholera virus (HOCV) and sheep border diseasevirus (BDV), in the pestivirus genus. Although Pestiviruses werepreviously classified in the Togaviridae family, some studies havesuggested their reclassification within the Flaviviridae family alongwith the flavivirus and hepatitis C virus (HCV) groups.

Equine herpesviruses (EHV) comprise a group of antigenically distinctbiological agents which cause a variety of infections in horses rangingfrom subclinical to fatal disease. These include Equine herpesvirus-1(EHV-1), a ubiquitous pathogen in horses. EHV-1 is associated withepidemics of abortion, respiratory tract disease, and central nervoussystem disorders. Other EHV's include EHV-2, or equine cytomegalovirus,EHV-3, equine coital exanthema virus, and EHV-4, previously classifiedas EHV-1 subtype 2.

Sheep and goats can be infected by a variety of dangerous microorganismsincluding visna-maedi.

Primates such as monkeys, apes and macaques can be infected by simianimmunodeficiency virus. Inactivated cell-virus and cell-free wholesimian immunodeficiency vaccines have been reported to afford protectionin macaques (Stott et al., Lancet 36:1538-1541 (1990); Desrosiers etal., PNAS USA 86:6353-6357 (1989); Murphey-Corb et al., Science246:1293-1297 (1989); and Carlson et al., AIDS Res. Human Retroviruses6:1239-1246 (1990)). A recombinant HIV gp120 vaccine has been reportedto afford protection in chimpanzees (Berman et al., Nature 345:622-625(1990)).

Cats, both domestic and wild, are susceptible to infection with avariety of microorganisms. For instance, feline infectious peritonitisis a disease which occurs in both domestic and wild cats, such as lions,leopards, cheetahs, and jaguars. When it is desirable to preventinfection with this and other types of pathogenic organisms in cats, themethods of the invention can be used to vaccinate cats to prevent themagainst infection.

Domestic cats may become infected with several retroviruses, includingbut not limited to feline leukemia virus (FeLV), feline sarcoma virus(FeSV), endogenous type C oncomavirus (RD-114), and felinesyncytia-forming virus (FeSFV). The discovery of feline T-lymphotropiclentivirus (also referred to as feline immunodeficiency) was firstreported in Pedersen et al., Science 235:790-793 (1987). Felineinfectious peritonitis (FIP) is a sporadic disease occurringunpredictably in domestic and wild Felidae. While FIP is primarily adisease of domestic cats, it has been diagnosed in lions, mountainlions, leopards, cheetahs, and the jaguar. Smaller wild cats that havebeen afflicted with FIP include the lynx and caracal, sand cat andpallas cat.

Viral and bacterial diseases in fin-fish, shellfish or other aquaticlife forms pose a serious problem for the aquaculture industry. Owing tothe high density of animals in the hatchery tanks or enclosed marinefarming areas, infectious diseases may eradicate a large proportion ofthe stock in, for example, a fin-fish, shellfish, or other aquatic life,forms facility. Prevention of disease is a more desired remedy to thesethreats to fish than intervention once the disease is in progress.Vaccination of fish is the only preventative method which may offerlong-term protection through immunity. Nucleic acid based vaccinationsof fish are described, for example, in U.S. Pat. No. 5,780,448.

The fish immune system has many features similar to the mammalian immunesystem., such as the presence of B cells, T cells, lymphokines,complement, and immunoglobulins. Fish have lymphocyte subclasses withroles that appear similar in many respects to those of the B and T cellsof mammals. Vaccines can be administered orally or by immersion orinjection.

Aquaculture species include but are not limited to fin-fish, shellfish,and other aquatic animals. Fin-fish include all vertebrate fish, whichmay be bony or cartilaginous fish, such as, for example, salmonids,carp, catfish, yellowtail, seabream and seabass. Salmonids are a familyof fin-fish which include trout (including rainbow trout), salmon andArctic char. Examples of shellfish include, but are not limited to,clams, lobster, shrimp, crab and oysters. Other cultured aquatic animalsinclude, but are not limited to, eels, squid and octopi.

Polypeptides of viral aquaculture pathogens include but are not limitedto glycoprotein or nucleoprotein of viral hemorrhagic septicemia virus(VHSV); G or N proteins of infectious hematopoietic necrosis virus(IHNV); VP1, VP2, VP3 or N structural proteins of infectious pancreaticnecrosis virus (IPNV); G protein of spring viremia of carp (SVC); and amembrane-associated protein, tegumin or capsid protein or glycoproteinof channel catfish virus (CCV).

Polypeptides of bacterial pathogens include but are not limited to aniron-regulated outer membrane protein, (IROMP), an outer membraneprotein (OMP), and an A-protein of Aeromonis salmonicida which causesfurunculosis, p57 protein of Renibacterium salmoninarum which causesbacterial kidney disease (BKD), major surface associated antigen (msa),a surface expressed cytotoxin (mpr), a surface expressed hemolysin(ish), and a flagellar antigen of Yersiniosis; an extracellular protein(ECP), an iron-regulated outer membrane protein (TROMP), and astructural protein of Pasteurellosis; an OMP and a flagellar protein ofVibrosis anguillarum and V. ordalii; a flagellar protein, an OMPprotein, aroA, and purA of Edwardsiellosis ictaluri and E. tarda; andsurface antigen of Ichthyophthirius; and a structural and regulatoryprotein of Cytophaga columnari; and a structural and regulatory proteinof Rickettsia.

Polypeptides of a parasitic pathogen include but are not limited to thesurface antigens of Ichthyophthirius.

In another aspect of the invention, there is provided vaccinecompositions suitable for use in methods for preventing and/orattenuating diseases or conditions which are caused or exacerbated by“self' gene products (e.g., tumor necrosis factors). Thus, vaccinecompositions of the invention include compositions which lead to theproduction of antibodies that prevent and/or attenuate diseases orconditions caused or exacerbated by “self' gene products. Examples ofsuch diseases or conditions include graft versus host disease, IgEmediated allergic reactions, anaphylaxis, adult respiratory distresssyndrome, Crohn's disease, allergic asthma, acute lymphoblastic leukemia(ALL), non Hodgkin's lymphoma (NHL), Graves' disease, systemic lupuserythematosus (SLE), inflammatory autoimmune diseases, myastheniagravis, immunoproliferative disease lymphadenopathy (IPL),angioimmunoproliferative lymphadenopathy (AIL), immunoblastivelymphadenopathy (IBL), rheumatoid arthritis, diabetes, prion diseases,multiple sclerosis, Alzheimer disease and osteoporosis.

In related specific embodiments, compositions of the invention are animmunotherapeutic that can be used for the treatment and/or preventionof allergies, cancer or drug addiction.

The selection of antigens or antigenic determinants for the preparationof compositions and for use in methods of treatment for allergies wouldbe known to those skilled in the medical arts treating such disorders.Representative examples of such antigens or antigenic determinantsinclude the following: bee venom phospholipase A2, Bet v I (birch pollenallergen), 5 Dol m V (white-faced hornet venom allergen), and Der p I(House dust mite allergen), as well as fragments of each which can beused to elicit immunological responses.

The selection of antigens or antigenic determinants for compositions andmethods of treatment for cancer would be known to those skilled in themedical arts treating such disorders (see Renkvist et al., Cancer.Immunol Immunother. 50:3-15 (2001) which is incorporated by reference),and such antigens or antigenic determinants are included within thescope of the present invention. Representative examples of such types ofantigens or antigenic determinants include the following: Her2 (breastcancer); GD2 (neuroblastoma); EGF-R (malignant glioblastoma); CEA(medullary thyroid cancer); CD52 (leukemia); human melanoma proteingp100; human melanoma protein gp100 epitopes such as amino acids 154-162(sequence: KTWGQYWQV) (SEQ ID NO: 63), 209-217 (ITDQVPFSV) (SEQ ID NO:64), 280-288 (YLEPGPVTA) (SEQ ID NO: 65), 457-466 (LLDGTATLRL) (SEQ IDNO: 66) and 476-485 (VLYRYGSFSV) (SEQ ID NO: 67); human melanoma proteinmelan-A/MART-1; human melanoma protein melan-A/MART-1 epitopes such asamino acids 26-35 (EAAGIGILTV) (SEQ ID NO:68), 27-35 (AAGIGILTV) (SEQ IDNO: 69) and 32-40 (ILTVILGVL) (SEQ ID NO: 70); tyrosinase and tyrosinaserelated proteins (e.g., TRP-1 and TRP-2); tyrosinase epitopes such asamino acids 1-9 (MLLAVLYCL) (SEQ ID NO: 71) and 369-377 (YMDGTMSQV) (SEQID NO: 72); NA17-A nt protein; NA17-A nt protein epitopes such as aminoacids 38-64 (VLPDVFIRC) (SEQ ID NO: 73); MAGE-3 protein; MAGE-3 proteinepitopes such as amino acids 271-279 (FLWGPRALV) (SEQ ID NO: 74); otherhuman tumors antigens, e.g. CEA epitopes such as amino acids 571-579(YLSGANLNL) (SEQ ID NO: 75); p53 protein; p53 protein epitopes such asamino acids 65-73 (RMPEAAPPV) (SEQ ID NO: 76), 149-157 (STPPPGTRV) (SEQID NO: 77) and 264-272 (LLGRNSFEV) (SEQ ID NO: 78); Her2/neu epitopessuch as amino acids 369-377 (KIFGSLAFL) (SEQ ID NO: 79) and 654-662(IISAVVGIL) (SEQ ID NO: 80); NY-ESO-1 peptides 157-165 and 157-167,159-167; HPV16 E7 protein; HPV16 E7 protein epitopes such as amino acids86-93 (TLGIVCPI) (SEQ ID NO: 81); as well as fragments or mutants ofeach which can be used to elicit immunological responses.

The natural MelanA/Mart-1 epitopes, and for example the MelanA/Mart-126-35 epitope bind with low affinity to human HLA-2 only. Thus, in vivopresentation of the natural MelanA epitopes and peptides, respectively,upon vaccination may be a limiting factor. This is particularlyimportant if Melan A epitopes and peptides, respectively, bound, coupledor fused to VLPs are used for vaccination, since under these conditions,MelanA peptides load HLA molecules by cross-presentation. The process ofcross-presentation is, however, not as efficient as classical pathwaysof antigen presentation and the affinity of the MelanA peptide for HLAis even more important. Thus, for VLP-based vaccinations, it is verypreferable to use MelanA peptides that bind with relatively highaffinity to HLA. Similarly, it may also be advantageous to use MelanApeptides that are recognized with higher affinity by the natural T cellrepertoire of the host. As a general rule, MelanA epitopes and peptides,respectively, are preferred that contain anchor residues at the properpositions allowing for efficient binding to MHC molecules.

Therefore, a further aspect of the present invention and a preferredembodiment of the present invention is to provide a composition forenhancing an immune response in an animal comprising (a) a virus-likeparticle; and (b) an immunostimulatory substance, wherein saidimmunostimulatory substance is bound to said virus-like particle, andwherein said composition further comprises at least one antigen orantigenic determinant, wherein said antigen or antigenic determinant isbound to said virus-like particle, and wherein said at least one antigenor antigenic determinant comprises, alternatively consists essentiallyof, or alternatively consists of a human melanoma MelanA peptideanalogue, and wherein said human melanoma MelanA peptide analogue isbound to said virus-like particle.

The term “natural human Melan A peptide” or “normal human Melan Apeptide” as used herein, shall refer to a peptide comprising, oralternatively consisting essentially of, or consisting of the amino acidsequence EAAGIGILTV (SEQ ID NO: 68) representing amino acids positions26-35 of the normal human MelanA protein sequence or AAGIGILTV (SEQ IDNO: 69) representing amino acids positions 27-35 of the normal humanMelanA protein sequence.

A “MelanA peptide analogue” as used herein shall be defined as a peptidein which the amino acid sequence of the corresponding naturuallyoccurring normal MelanA peptide is altered by at least one amino acid oramino acid derivative, wherein this alteration may comprise an aminoacid substitution and/or deletion and/or insertion or a combinationthereof. In a preferred embodiment of the present invention, the term“MelanA peptide analogue A” as used herein shall be defined as a peptidein which the amino acid sequence of the corresponding naturuallyoccurring normal MelanA peptide is altered by three, preferably two, andeven more preferably one, amino acid or amino acid derivative, whereinthis alteration may comprise an amino acid substitution and/or deletionand/or insertion or a combination thereof. In a further preferredembodiment of the present invention, the term “MelanA peptide analogueA” as used herein shall be defined as a peptide in which the amino acidsequence of the corresponding naturually occurring normal MelanA peptideis altered by three, preferably two, and even more preferably one, aminoacid or amino acid derivative, wherein this alteration may comprise anamino acid substitution and/or deletion and/or insertion or acombination thereof, and wherein this alteration is at position 26, 27,28 and/or 35 of the normal human MelanA protein sequence (SEQ ID NO:109).

In a preferred embodiment of the present invention, the Melan A peptideanalogue is capable of allowing an efficient binding to MHC molecules.The use of a MelanA peptide analogue, thus, allows, in particular, theintroduction of such anchor residues leading to an improved binding toMHC molecules. The introduction of such anchor residues leading to animproved binding to MHC molecules is in particular advantageous, if thenatural and normal, respectively, MelanA peptide does not contain suchanchor residues or does not contains only such anchor residues which areinferior to the newly introduced anchor residue(s) replacing the naturaland normal, respectively anchor residue.

The modification of the normal human MelanA peptide leading to theMelanA peptide analogue, and hereby preferably the introduction of theseanchor residues is effected either by (i) induced mutation (e.g.chemical induction, irradiation or other procedures known to a personskilled in the art) and subsequent selection of modified peptides withimproved binding to MHC or (ii) of selection of modified peptides withimproved binding to MHC arising from natural mutations arising at anylevel of protein synthesis, including but not limited to mutationsarising at the DNA, transcriptional, RNA or translational level ofprotein expression or (iii) or by systematic or random amino acidexchanges, deletions, substitutions or insertions by using classicalpeptide synthesis known by the person skilled in the art. Theidentification of such anchor residues is typically and preferablyeffected by using the SYFPEITHI database as described by Rammensee etal. in Immunogenetics 50:213-219 (1999). The SYFPEITHI database allowscalculating the efficiency of HLA binding for any peptide of choice andit is possible to optimize the peptides regarding the efficiency of HLAbinding using this program. Alternatively, identification of preferredpeptide analogues can be achieved by MHC-peptide binding assaysinvolving but not limited to whole cell assays of T cell activation orrecognition or WIC upregualtion in mutant cell lines,MHC-tetramer-peptide binding assays, competitive binding assays withlabelled peptides, surface plasmon resonance assays, all known to theperson skilled in the art.

In a further preferred embodiment of the present invention, the MelanApeptide analogue is characterized by two, more preferably by a singleamino acid substitution with respect to the corresponding normal MelanApeptide.

In another preferred embodiment of the present invention, the MelanApeptide analogue is protected from protease or peptidase mediateddegradation. The use of MelanA peptide analogues that are protected fromprotease or peptidase degradation leads to increased stability of thepeptide in vivo after application of the peptide to a subject or and/orto increased stability of the peptide during storage in the presence ofproteases or peptidases. The consequence of this increased stability ismore efficient and prolonged presentation of the human melanoma MelanApeptide analogue on MHC and thus the enhanced stimulation of a specificT cell response.

Preferably, the human MelanA peptide analogue is protected bysubstitution of selected amino acid residues of the natural human MelanApeptide by non natural amino acid derivatives as exemplified in Blanchetet al, J. Immunol. 167:5852-5861 (2001) and references cited therein.This overcomes the limitation typically imposed by the fact thatchemically modified MelanA peptides and MelanA peptide analogues,respectively, may not be recognized by the T cells equally well ascompared to the natural and normal, respectively, MelanA peptide.

In another preferred embodiment, the antigen comprises, alternativelyconsists essentially of, or alternatively consists of a human melanomaMelanA/MART-1 peptide analogue having an amino acid sequence which isselected from the group consisting of (a) LAGIGILTV (SEQ ID NO: 89); (b)MAGIGILTV (SEQ ID NO: 90); (c) EAAGIGILTV (SEQ ID NO: 68), (d)EAMGIGILTV (SEQ ID NO: 91), (e) ELAGIGILTV (SEQ ID NO: 35), (f)EMAGIGILTV (SEQ ID NO: 92), (g) YAAGIGILTV (SEQ ID NO: 93), and (h)FAAGIGILTV (SEQ ID NO: 94). These peptide analogues as well as theirsynthesies have been described by Valmori at al., J. Immunol. 160:1750-1758 (1998). These peptide analogues show increased relativerecognition and target cell lysis by five different cytotoxic T cellclones raised against the natural melanoma peptide.s

In a very preferred embodiment of the present invention the humanmelanoma MelanA/MART-1 peptide analogue comprises, alternativelyconsists essentially of, or alternatively consists of the sequenceELAGIGILTV (SEQ ID NO: 35). As indicated throughout the examples thisvery preferred embodiment induces expansion of functionalMelanA-specific CD8+T cells in HLA-A2 transgenic mice and represents agood compromise between HLA-binding and TCR-recognition (cf. Valmori atal., J. Immunol. 160: 1750-1758 (1998)).

In a further very preferred embodiment of the present invention thehuman melanoma MelanA/MART-1 peptide analogue with the second attachmentsite has an amino acid sequence selected from (a) CGHGHSYTTAEELAGIGILTV(SEQ ID NO: 40); and typically abbreviated herein as MelanA 16-35 A/L),(b) CGGELAGIGILTV (SEQ ID NO: 42); and typically abbreviated herein asMelanA 26-35 A/L), (c) CSYTTAEELAGIGILTVILGVL (SEQ ID NO: 43); andtypically abbreviated herein as MelanA 20-40 A/L), (d)CGGELAGIGILTVILGVL (SEQ ID NO: 44); and typically abbreviated herein asMelanA 26-40 A/L), (e) ELAGIGILTVGGC (SEQ ID NO: 45); typicallyabbreviated herein as MelanA 26-35-C A/L), (f) CSPKSLELAGIGILTV (SEQ IDNO: 77), and typically abbreviated herein as CSPKSL-MelanA 26-35 A/L;and (g) ELAGIGILTVILGVLGGC (SEQ ID NO: 78), and typically abbreviatedherein as MelanA 26-40-C A/L.

In another very preferred embodiment of the present invention the humanmelanoma MelanA/MART-1 peptide analogue with the second attachment hasan amino acid sequence of CGHGHSYTTAEELAGIGILTV (SEQ ID NO: 40).

The selection of antigens or antigenic determinants for compositions andmethods of treatment for drug addiction, in particular recreational drugaddiction, would be known to those skilled in the medical arts treatingsuch disorders. Representative examples of such antigens or antigenicdeterminants include, for example, opioids and morphine derivatives suchas codeine, fentanyl, heroin, morphium and opium; stimulants such asamphetamine, cocaine, MDMA (methylenedioxymethamphetamine),methamphetamine, methylphenidate and nicotine; hallucinogens such asLSD, mescaline and psilocybin; as well as cannabinoids such as hashishand marijuana.

The selection of antigens or antigenic determinants for compositions andmethods of treatment for other diseases or conditions associated withself antigens would be also known to those skilled in the medical artstreating such disorders. Representative examples of such antigens orantigenic determinants are, for example, lymphotoxins (e.g. Lymphotoxinα (LT α), Lymphotoxin β (LT β)), and lymphotoxin receptors, Receptoractivator of nuclear factor kappaB ligand (RANKL), vascular endothelialgrowth factor (VEGF) and vascular endothelial growth factor receptor(VEGF-R), Interleukin 17 and amyloid beta peptide (Aβ1-42), TNFα, MIF,MCP-1, SDF-1, Rank-L, M-CSF, Angiotensin II, Endoglin, Eotaxin, Grehlin,BLC, CCL21, IL-13, IL-17, IL-5, IL-8, IL-15, Bradykinin, Resistin, LHRH,GHRH, GIH, CRH, TRH and Gastrin, as well as fragments of each which canbe used to elicit immunological responses.

In a particular embodiment of the invention, the antigen or antigenicdeterminant is selected from the group consisting of: (a) a recombinantpolypeptide of HIV; (b) a recombinant polypeptide of Influenza virus(e.g., an Influenza virus M2 polypeptide or a fragment thereof); (c) arecombinant polypeptide of Hepatitis C virus; (d) a recombinantpolypeptide of Hepatitis B virus (e) a recombinant polypeptide ofToxoplasma; (f) a recombinant polypeptide of Plasmodium falciparum; (g)a recombinant polypeptide of Plasmodium vivax; (h) a recombinantpolypeptide of Plasmodium ovale; (i) a recombinant polypeptide ofPlasmodium malariae; (j) a recombinant polypeptide of breast cancercells; (k) a recombinant polypeptide of kidney cancer cells; (l) arecombinant polypeptide of prostate cancer cells; (m) a recombinantpolypeptide of skin cancer cells; (n) a recombinant polypeptide of braincancer cells; (o) a recombinant polypeptide of leukemia cells; (p) arecombinant profiling; (q) a recombinant polypeptide of bee stingallergy; (r) a recombinant polypeptide of nut allergy; (s) a recombinantpolypeptide of pollen; (t) a recombinant polypeptide of house-dust; (u)a recombinant polypeptide of cat or cat hair allergy; (v) a recombinantprotein of food allergies; (w) a recombinant protein of asthma; (x) arecombinant protein of Chlamydia; and (y) a fragment of any of theproteins set out in (a) (x).

In a further embodiment of the invention, the antigen or antigenicdeterminant is a polypeptide, a polyprotein, a peptide, an epitope or apolyepitope of HIV. Said polypeptide, polyprotein, peptide, epitope orpolyepitope of HIV is fused, coupled, bound or otherwise attached to theVLP or packaged VLP as set out throughout the present application, andleading to preferred embodiments of the invention.

HIV is a retrovirus and belongs to the family of the lentiviruses. Twotypes of HIV viruses have been discovered, HIV-1 and HIV-2. HIV-2 ismainly found in the countries of Western Africa, while HIV-1 is the mostcommon form of HIV elsewhere.

The overall structure of the HIV virus as well as of a number of itscomponents are well known, although no crystal structure of the wholevints is available yet (Turner, B. G. et al., J. Mol. Biol. 285: 1-32(1999)). There is strong evidence for a central role of HIV specificT-cells in controlling HIV viral replication (Jin X., et al., J. Exp.Med. 189: 1365-1372 (1999)). There have been numerous attempts todevelop vaccination strategies eliciting T-cell responses against HIV,and in particular cytotoxic T-cell (CTL) responses. Those approacheshave so far worked nicely in murine and non-human primate models, butare significantly less effective in humans (Moingeon P. et al., J.Biotechnol. 98: 189-198 (2002)). DNA vaccination, use of non replicatingadenoviral vector (Shiver, J. W. et al., Nature 415:331-335 (2002)), orlive attenuated viruses (Ranke, T. et al., Nat. Med. 6: 951-955 (2000))have been described. Combination of two of those approaches in a socalled prime boost regimen has also been described (Allen, T. M. et al.,J. Immunol. 164: 4968-4978 (2000)). These approaches however suffer froma number of disadvantages. DNA immunisation may lead to integration ofDNA into the genome, plasmid DNA may contain resistance genes, viralpromoters are used, and antibodies to DNA may be elicited in the host.Furthermore, large amounts of DNA are required. The use of liveattenuated or replication deficient viruses always bears the risk ofrecombination, which might lead to more virulent species, which is aconcern particularly in immunocompromised individuals. The use of viralvectors is expected to lead to the infection of a large number ofdifferent cell types in the body, and indeed infection is required forthe efficacy of the vaccine. Finally, the use of adenoviral vectors maybe inefficient or lead to side effects in patients sero-positive foradenovirus. There is therefore a need for a safe and immunogenic vaccinetechnology to induce strong and potent CTL responses against HIV.

Therefore, a further aspect of the present invention and a preferredembodiment of the present invention is to provide a composition forenhancing an immune response in an animal comprising: (a) a virus-likeparticle; (b) an immunostimulatory substance; and (c) at least oneantigen or antigenic determinant; wherein said immunostimulatorysubstance is bound to said virus-like particle, and wherein said antigencomprises, alternatively consists essentially of, or alternativelyconsists of at least one HIV polypeptide, and wherein said at least oneHIV polypeptide is bound to said virus-like particle.

A “HIV polypeptide” as used herein shall include a polypeptide, apolyprotein, a peptide, an epitope of HIV. In a preferred embodiment ofthe present invention the term “HIV polypeptide” as used herein shallrefer to a polypeptide of HIV comprising, or alternatively consistingessentially of, or alternatively consisting of an epitope of HIV. In afurther preferred embodiment of the present invention, the antigen orantigenic determinant comprises, or alternatively consists essentiallyof, or alternatively consists of a polyepitope of HIV. The term“polyepitope of HIV” as used herein shall refer to a combination of atleast two HIV polypeptides, wherein said at least two HIV polypeptidesare bound directly or by way of a linking sequence.

In a very preferred embodiment of the present invention the antigencomprises, or alternatively consists essentially of, or alternativelyconsists of is a combination of at least two HIV polypeptides, whereinsaid at least two HIV polypeptides are bound directly or by way of alinking sequence.

VLPs bound, coupled, or otherwise fused to HIV antigens are particularlysuited as a safe, non-infectious and non-replicative vaccine to induceT-cells and in particular CTLs against HIV. VLPs are particularlyeffective when they are packaged with immunostimulatory substances andsequences, respectively. The use of a defined vaccine and thus defineddoses of antigen is another advantage over the use of viral vectors,where the antigen dose is more difficult to evaluate. Finally, VLPstarget preferentially dendritic cells and macrophages (Ruedl, C. et al.,Eur. J. Immunol. 32: 818-825 (2002)), ensuring antigen delivery to themost relevant antigen presenting cells. VLP based vaccines havetherefore a much higher specificity than viral-vector or DNA basedvaccines.

Suitable HIV antigens and poylpetides, respectively, for preparation ofthe compositions of the invention include the following HIV proteinsubunits: p17-GAG, p24-GAG, p15-GAG, Protease, reverse transcriptase(RT), Integrase, Vif, Vpr, Vpu, Tat, Rev, gp-41-Env, gp-120-Env and Nef(Addo, M. M. et al., J. Virol. 77: 2081-2092 (2003)). Both the wholeprotein subunits and fragments thereof are suitable in preparing thecompositions of the invention. In particular, chemically synthesizedpeptides having the sequence of fragments of these subunits are alsoincluded. Polyepitopes, which may be obtained as recombinantpolypeptides or as chemically synthesized long peptides, are used in afavored embodiment of the invention for binding, coupling or otherwiseattachment to the VLP and preferably packaged VLP. The DNA sequenceencoding a polyepitope may also be fused in frame to the sequence of aVLP subunit, leading to VLPs or packaged VLPs fused to the polyepitope.In the case where the HIV antigen is coupled to the VLP using across-linker containing a maleimide moiety, the HIV antigen, a peptideor recombinant polypeptide, is modified according to the disclosures ofthe invention to include a cysteine residue for reaction with themaleimide moiety introduced in the VLP after the derivatization step ofthe cross-linking procedure.

A prominent feature of HIV infection, is the ability of the virus toescape from immune control, through accumulation of mutations which areselected for by the strong CTL response elicited in the host (McMichael,A. J. & Rowland-Jones, S. L. Nature 410: 980-987 (2001)). It istherefore advantageous to immunize and induce T-cells against adiversity of epitopes, in order to limit the effect of mutations insingle epitopes. A composition of the invention suitable for eliciting aT-cell response against a plurality of epitope will for example beprepared by coupling at least two, or alternatively a plurality ofepitopes, in the form of chemically synthesized peptides modifiedaccordingly for cross-linking, to a VLP or packaged VLP. As a result,VLPs or packaged VLPs each coupled to at least two, or alternativelyseveral different HIV polypeptides and therefore epitopes are obtained.In another approach, a peptide and polypeptide, respectively, containingat least two, or alternatively several consecutive HIV epitopes eitheroriginating from the same or from different HIV antigens, i.e. apreferred polyepitope of HIV for the present invention, is coupled,bound, fused or otherwise attached to a VLP or packaged VLP. Likewise,at least two, or alternatively several different polyepitopes may alsobe coupled, fused or otherwise attached to one VLP or packaged VLP. Inyet another embodiment of the invention, at least two, or alternativelyseveral different HIV antigens, in the form of recombinant polypeptides,are coupled or bound to one VLP or packaged VLP. Alternatively, apolyprotein, that is a fusion protein comprising two or more HIVpolypeptides, modified according to the disclosures of the presentinvention for coupling, binding or fusion to a VLP, is used as antigenor antigenic determinant. In a further embodiment, combination ofpeptides, polyepitopes and recombinant polypeptides are coupled, boundor otherwise attached to one VLP or packaged VLP. In a yet furtherembodiment of the invention, the HIV antigens are fused to one VLP orpackaged VLP.

Immunisation of an animal or subject with a plurality of HIV antigens isalso achieved in one further embodiment of the invention by mixingdifferent particles, each coupled, bound, fused or otherwise attached toone, two or more HIV antigens, said HIV antigens being a peptide, anepitope a recombinant polypeptide or a polyepitope. As HTV virus isconstantly mutating, it has been recognized that the sequence of theantigens of a given HIV primary isolate may be more remote in sequenceidentity from the sequences of so called autologuous viruses present ina given population, than a consensus sequence built from the sequencesavailable in the database (The Identification of Optimal HIV-Derived CTLEpitopes in Diverse Populations Using HIV Clade-Specific Consensus, pp.I-1-20 in HIV Molecular Immunology 2001. Edited by: Korber BTK, BranderC, Haynes B F, Koup R, Kuiken C, Moore J P, Walker B D, and Watkins D,Published by: Theoretical Biology and Biophysics Group, Los AlamosNational Laboratory, Los Alamos, N.Mex., LA-UR 02-2877). The sequencesof epitopes to be coupled, fused, bound or otherwise attached to a VLPor packaged VLP as peptide, polyepitope or included in a recombinantpolypeptide or polyprotein are therefore preferably consensus sequences,obtained from the database (see above reference, or website:http://hiv-web.lanl.gov/seq-db.html) or obtained by aligning allsequences of a given antigen from the database. In preferredembodiments, sequences from one clade of virus are selected, in functionof the most prevalent clade in the geographical region where thecompositions of the invention or vaccines are intended to be injected.Aligning sequences of the database would be known to one skilled in theart. For example, the program Blast (Altschul, S. F et al., J. Mol.Biol. 215:403-410 (1990); Altschul, S. F. et al., Nature Genet.6:119-129 (1994)) or FASTA (Pearson, W. R. Methods Enzymol. 183:63-98(1990)) may be used to perform the sequence alignments.

The HIV antigens p24-GAG and Nef have been found to have the highestepitope density (Addo, M. M. et al., J. Virol. 77: 2081-2092 (2003)). Inpreferred embodiments of the invention, the antigen or antigenicdeterminant comprises therefore p24-GAG-CTL and/or NEF-CTL and/or Thcell epitopes. Th cell epitopes are believed to contribute to theinduction and maintenance of CTL responses, and therefore, in preferredembodiments of the invention, Th cell epitopes are included in thecomposition of the invention. For example, Th cell epitopes may beincluded in a polyepitope or polyprotein. Alternatively, peptidescomprising Th cell epitopes may be coupled to VLPs or packaged VLPs, orthe composition of the invention may be a mixture of particles, eachcoupled to an individual peptide, and one or more of said peptides maycomprise one or more Th cell epitopes.

In very preferred embodiments of the invention, the antigen or antigenicdeterminant with the second attachment site is selected from the groupof the GAG polyepitopes gag-G50 (SEQ ID NO: 86), gag-G68n (SEQ ID NO:88) and of the Nef polyepitope nef-N56 (SEQ ID NO: 87). Gag-50, gag-68nand nef-N56 comprise polyepitopes derived from the Clade B consensussequences of gag and nef (The Identification of Optimal HIV-Derived CTLEpitopes in Diverse Populations Using HIV Clade-Specific Consensus, pp.I-1-20 in HIV Molecular Immunology 2001. Edited by: Korber B T K,Brander C, Haynes B F, Koup R, Kuiken C, Moore J P, Walker B D, andWatkins D. Published by: Theoretical Biology and Biophysics Group, LosAlamos National Laboratory, Los Alamos, N.Mex., LA-UR 02-2877; onlinedatabase on HIV epitopes and consensus sequence,http://hiv-web.lanl.gov/seq-db.html).

The nef-N56 polyepitope, starting with the aminoacid number 66 of theNef-protein consensus sequence (SEQ ID NO: 96), comprises amino acids66-99 (VGFPVRPQVPLRPMTYKAAVDLSHFLKEKGGLEG, (SEQ ID NO: 98), followed byamino acids 131-150 (PGIRYPLTFGWCFKLVPVEP, (SEQ ID NO: 99) of the HIV-1clade B Nef-protein consensus sequence (SEQ ID NO: 96). The resultingpolypeptide, i.e. the combination of SEQ ID NQ: 98 and SEQ ID NO: 99,has the amino acid sequence of SEQ ID NO: 104. The nef-N56 polyepitopeadditionally comprises an N-terminal Cysteine and Glycine for coupling(SEQ ID NO: 87).

The gag-G50 polyepitope starts at the N-terminus of p24-GAG, fromposition 139 of the HIV-1 clade B GAG-protein consensus sequence (SEQ IDNO: 97). The sequence “KVVEE” ((SEQ ID NO: 100) which represents theamino acids 157-161 from the GAG consensus sequence (SEQ ID NO: 97)),and where the density of epitopes is lowest, is deleted. Thus, gag-G50comprises amino acids 139-156 (QGQMVHQAISPRTLNAWV, (SEQ ID NO: 101)),followed by amino acids 162-191 (KAFSPEVIPMFSALSEGATPQDLNTMLNTV (SEQ IDNO: 102)) of the GAG-protein consensus sequence (SEQ ID NO: 97). Theresulting polypeptide, i.e. the combination of SEQ ID NO: 101 and SEQ IDNO: 102, has the amino acid sequence of SEQ ID NO: 105. In a preferredembodiment, the gag-G50 polyepitope comprises an N-terminal Cysteine forcoupling (SEQ ID NO: 106). In another preferred embodiment, inparticular to improve solubility, the gag-G50 polyepitope additionallycomprises a C-terminal lysine residue (SEQ ID NO: 86).

The gag-G68n epitope (SEQ ID NO: 88) is based on G50 epitope, with theaddition of the more C-terminal “GEIYKRWIILGLNKIVRMY” sequence,corresponding to aminoacids 259-277 (SEQ ID NO: 103) from GAG-proteinconsensus sequence (SEQ ID NO: 97) to the N-terminus of the sequence ofgag-G50 (excluding the N-terminal cysteine). Therefore, the resultingHIV polypeptide, i.e. the combination of SEQ ID NO: 103, SEQ ID NO: 101and SEQ ID NO: 102, has the amino acid sequence of SEQ ID NO: 172. In apreferred embodiment, the gag-G68n epitope comprises an N-terminalCysteine for coupling (SEQ ID NO: 108). In another preferred embodiment,in particular to improve solubility, the gag-G68n epitope additionallycomprises a C-terminal lysine residue (SEQ ID NO: 88).

In a preferred embodiment, the polyepitopes of the invention comprise acysteine residue at the N-terminus for coupling, rather than aC-terminal cysteine, since there are more protecting strategies forN-terminal cysteines, and peptides may be further trimmed at theirN-terminus for proper presentation by aminopeptidases (Goldberg A. L. etal., Mol. Immunol. 39: 147-164 (2002)). Introduction of the cysteineresidue for coupling to the C-terminus rather than the N-terminushowever also leads to an embodiment of this invention.

In further preferred embodiments of the invention, the polyepitopesgag-G50 (SEQ ID NO: 86), nef-N56 (SEQ ID NO: 87) or gag-G68n (SEQ ID NO:88) are coupled to the RNA phage VLPs or packaged VLPs Qβ, AP205, GA,MS-2 and fr, or to HBcAg VLPs or packaged VLPs modified to harbour anadditional lysine residue in their immunodominant region, i.e.HBcAg1-185lys described in WO 02/56905 which is incorporated hereby inits entirety by way of reference. In a further preferred embodiment ofthe invention, the two polyepitopes gag-G50 and nef-N56 are coupled bothon a single VLP. In a yet further embodiment of the invention, the VLPis the VLP of RNA phages Qβ, AP205, GA, MS-2 and Fr, or HBcAg1-185lysbeing described in WO 02/56905 which is incorporated hereby in itsentirety by way of reference.

In specific embodiments of the invention, the gag-G50 and gag-G68n, andthe nef-N56 epitopes are fused to the N-terminus of the VLP of phage fr,or to the C-terminus of phage Qβ.

Expression and purification of the GAG protein (Berthet-Colominas, C. etal., EMBO J. 18: 1124-1136 (1999))), and the Nef protein or proteinfragments (Franken, P. et al., Prot. Sci. 6: 2681-2683 (1997)) of HIVhave been described, and in a further embodiment of the invention, GAGand NEF proteins, or fragments thereof, modified to include a cysteineresidue for coupling according to the disclosure of the presentinvention, are coupled to VLPs or packaged VLPs.

The compositions of the invention comprising a polypeptide, apolyprotein, a peptide, an epitope or a polyepitope of HIV andoptionally a further adjuvant, are useful as vaccines for induction ofHIV specific T-cells in humans. In a preferred embodiment of theinvention, the vaccine comprises a Qβ or AP205 VLP packaged with theG8-8 oligodeoxynucleotide and optionally a further adjuvant. The T-cellresponse induced upon vaccination is assessed in proliferation assays(for Th cell response, Belshe R. B. et al., J. Inf. Dis. 183: 1343-1352(2001)), in ELISPOT assays (Oxenius, A. et al., Proc. Natl. Acad. Sci.USA 99: 13747-13752 (2002)), or in Cytotoxicity assays (Belshe R. B. etal., J. Int. Dis. 183: 1343-1352 (2001)).

In a further embodiment, gag-G50, gag-G68n and nef-N56 devoid of theN-terminal cysteine are inserted between amino acid 2 and 3 (numberingof the cleaved CP, that is wherein the N-terminal methionine is cleaved)of the fr CP. In a related embodiment of the invention, gag-G50,gag-G68n and nef-N56 devoid of the N-terminal cysteine are fused to theA1 protein of Qβ VLP, as described above.

In another embodiment of the present invention, the antigen, beingcoupled, fused or otherwise attached to the virus-like particle, is a Tcell epitope, either a cytotoxic or a Th cell epitope. In a furtherpreferred embodiment, the antigen is a combination of at least two,preferably different, epitopes, wherein the at least two epitopes arelinked directly or by way of a linking sequence. These epitopes arepreferably selected from the group consisting of cytotoxic and Th cellepitopes.

It should also be understood that a mosaic virus-like particle, e.g. avirus-like particle composed of subunits attached to different antigensand epitopes, respectively, is within the scope of the presentinvention. Such a composition of the present invention can be, forexample, obtained by transforming E. coli with two compatible plasmidsencoding the subunits composing the virus-like particle fused todifferent antigens and epitopes, respectively. In this instance, themosaic virus-like particle is assembled either directly in the cell orafter cell lysis. Moreover, such an inventive composition can also beobtained by attaching a mixture of different antigens and epitopes,respectively, to the isolated virus-like particle.

The antigen of the present invention, and in particular the indicatedepitope or epitopes, can be synthesized or recombinantly expressed andcoupled to the virus-like particle, or fused to the virus-like particleusing recombinant DNA techniques. Exemplary procedures describing theattachment of antigens to virus-like particles are disclosed in WO00/32227, in WO 01/85208 and in WO 02/056905, the disclosures of whichare herewith incorporated by reference in its entirety.

The invention also provides a method of producing a composition,typically and preferably for enhancing an immune response in an animal,comprising a VLP and an immunostimulatory substance, preferably anunmethylated CpG-containing oligonucleotide bound to the VLP whichcomprises incubating the VLP with the immunostimulatory substance andoligonucleotide, respectively, adding RNase and purifying saidcomposition, wherein preferably the immunostimulatory substance is anunmethylated CpG-containing oligonucleotide, wherein the CpG motif ofthe unmethylated CpG-containing oligonucleotide is part of a palindromicsequence, and wherein the palindromic sequence is flanked at its3′-terminus and at its 5′-terminus by less than 10 guanosine entities.Preferably, the method further comprises the step of binding an antigenor antigenic determinant to said virus-like particle. In a preferredembodiment, the anigen or antigenic determinant is bound to thevirus-like particle before incubating the virus-like particle with theimmunostimulatory substance. In another preferred embodiment, the anigenor antigenic determinant is bound to the virus-like particle afterpurifying the composition. In an equally preferred embodiment, themethod comprises incubating the VLP with RNase, adding theimmunostimulatory substance and oligonucleotide, respectively, andpurifying the composition, wherein preferably the immunostimulatorysubstance is an unmethylated CpG-containing oligonucleotide, wherein theCpG motif of the unmethylated CpG-containing oligonucleotide is part ofa palindromic sequence, and wherein the palindromic sequence is flankedat its 3′-terminus and at its 5′-terminus by less than 10 guanosineentities. Preferably, the method further comprises the step of bindingan antigen or antigenic determinant to said virus-like particle. In apreferred embodiment, the anigen or antigenic determinant is bound tothe virus-like particle before incubating the virus-like particle withthe RNase. In another preferred embodiment, the anigen or antigenicdeterminant is bound to the virus-like particle after purifying thecomposition. In one embodiment, the VLP is produced in a bacterialexpression system. In another embodiment, the RNase is RNase A.

The invention further provides a method of producing a composition forenhancing an immune response in an animal comprising a VLP bound to animmunostimulatory substance, preferably to an unmethylatedCpG-containing oligonucleotide which comprises disassembling the VLP,adding the immunostimulatory substance and oligonucleotide,respectively, and reassembling the VLP, wherein preferably theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of the unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence, andwherein the palindromic sequence is flanked at its 3′-terminus and atits 5′-terminus by less than 10 guanosine entities. The method canfurther comprise removing nucleic acids of the disassembled VLP and/orpurifying the composition after reassembly. Preferably, the methodfurther comprises the step of binding an antigen or antigenicdeterminant to the virus-like particle. In a preferred embodiment, theanigen or antigenic determinant is bound to the virus-like particlebefore disassembling the virus-like particle. In another preferredembodiment, the anigen or antigenic determinant is bound to thevirus-like particle after reassembling the virus-like particle andpreferably after purifying the composition.

The invention also provides vaccine compositions which can be used forpreventing and/or attenuating diseases or conditions. Vaccinecompositions of the invention comprise, or alternatively consist of, animmunologically effective amount of the inventive immune enhancingcomposition together with a pharmaceutically acceptable diluent, carrieror excipient. The vaccine can also optionally comprise an adjuvant.

Thus, in a preferred embodiment, the invention provides a vaccinecomprising an immunologically effective amount of the inventive immuneresponse enhancing composition together with a pharmaceuticallyacceptable diluent, carrier or excipient, wherein the compositioncomprises, (a) a virus-like particle; (b) at least one immunostimulatorysubstance; and (c) at least one antigen or antigenic determinant;wherein the antigen or antigenic determinant is bound to the virus-likeparticle, and wherein the immunostimulatory substance is bound to thevirus-like particle, and wherein the antigen comprises, alternativelyconsists essentially of, or alternatively consists of a human melanomaMelanA peptide analogue. Preferably, the vaccine further comprises anadjuvant.

The invention further provides vaccination methods for preventing and/orattenuating diseases or conditions in animals. In one embodiment, theinvention provides vaccines for the prevention of infectious diseases ina wide range of animal species, particularly mammalian species such ashuman, monkey, cow, dog, cat, horse, pig, etc. Vaccines can be designedto treat infections of viral etiology such as HIV, influenza, Herpes,viral hepatitis, Epstein Bar, polio, viral encephalitis, measles,chicken pox, etc.; or infections of bacterial etiology such aspneumonia, tuberculosis, syphilis, etc.; or infections of parasiticetiology such as malaria, trypanosomiasis, leishmaniasis,trichomoniasis, amoebiasis, etc.

In another embodiment, the invention provides vaccines for theprevention of cancer in a wide range of species, particularly mammalianspecies such as human, monkey, cow, dog, cat, horse, pig, etc. Vaccinescan be designed to treat all types of cancer including, but not limitedto, lymphomas, carcinomas, sarcomas and melanomas.

It is well known that homologous prime-boost vaccination strategies withproteins or viruses are most often unsuccessful. Preexisting antibodies,upon re-encountering the antigen, are thought to interfere with theinduction of a memory response. To our surprise, the RNA-phage derivedVLPs, in particular the VLP derived from Qβ, do very efficiently inducea memory CD8⁺ T cell response in a homologous prime-boost vaccinationscheme. In contrast, as described in Example 29, live vaccinia virusimmunizations are very ineffective for the induction of a primary CD8⁺ Tcell response and homologous boosting with vaccinia does hardly lead toan expansion of memory CD8⁺ T cells.

Therefore, in a further aspect, the invention provides a method ofimmunizing or treating an animal comprising priming a T cell response inthe animal by administering an immunologically effective amount of theinventive vaccine. Preferably, the method further comprises the step ofboosting the immune response in the animal, wherein preferably theboosting is effected by administering an immunologically effectiveamount of a vaccine of the invention or an immunologically effectiveamount of a heterologous vaccine, wherein even more preferably theheterologous vaccine is a DNA vaccine, peptide vaccine, recombinantvirus or a dendritic cell vaccine.

Moreover, in again another aspect, the invention further provides amethod of immunizing or treating an animal comprising the steps ofpriming a T cell response in the animal, and boosting a T cell responsein the animal, wherein the boosting is effected by administering animmunologically effective amount of the vaccine of the invention.Preferably, the priming is effected by administering an immunologicallyeffective amount of a vaccine of the invention or an immunologicallyeffective amount of a heterologous vaccine, wherein even more preferablysaid heterologous vaccine is a DNA vaccine, peptide vaccine, recombinantvirus or a dendritic cell vaccine.

Moreover, in again another aspect, the invention further provides for acomposition comprising a virus-like particle, at least oneimmunostimulatory substance; and at least one antigen or antigenicdeterminant; wherein said antigen or antigenic determinant is bound tosaid virus-like particle, and wherein said immunostimulatory substanceis bound to said virus-like particle, and wherein said antigen comprisesa cytotoxic T cell epitope, a Th cell epitope or a combination of atleast two of said epitopes, wherein said at least two epitopes are bounddirectly or by way of a linking sequence, and wherein preferably saidcytotoxic T cell epitope is a viral or a tumor cytotoxic T cell epitope.

In again a further aspect, the present invention provides a composition,typically and preferably for enhancing an immune response in an animalcomprising: (a) a virus-like particle; (b) an immunostimulatorysubstance; wherein said immunostimulatory substance (b) is bound to saidvirus-like particle (a); and (c) an antigen, wherein said antigen ismixed with said virus-like particle (a), and wherein saidimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence, andwherein said palindromic sequence is flanked at its 3′-terminus and atits 5′-terminus by less than 10 guanosine entities. As used herein, theterm “mixed” refers to the combination of two or more substances,ingredients, or elements that are added together, are not chemicallycombined with each other and are capable of being separated. Methods ofmixing antigens with virus-like particles are described in WO 04/000351,which is incorporated herein by reference in its entirety.

As would be understood by one of ordinary skill in the art, whencompositions of the invention are administered to an animal, they can bein a composition which contains salts, buffers, adjuvants or othersubstances which are desirable for improving the efficacy of thecomposition. Examples of materials suitable for use in preparingpharmaceutical compositions are provided in numerous sources includingREMINGTON'S PHARMACEUTICAL SCIENCES (Osol, A, ed., Mack Publishing Co.,(1990)).

Various adjuvants can be used to increase the immunological response,depending on the host species, and include but are not limited to,Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette Guerin) and Corynebacterium parvum. Suchadjuvants are also well known in the art. Further adjuvants that can beadministered with the compositions of the invention include, but are notlimited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS 21,QS 18, CRL1005, Aluminum salts, MF 59, and Virosomal adjuvanttechnology. The adjuvants can also comprise a mixture of thesesubstances.

Compositions of the invention are said to be “pharmacologicallyacceptable” if their administration can be tolerated by a recipientindividual. Further, the compositions of the invention will beadministered in a “therapeutically effective amount” (i.e., an amountthat produces a desired physiological effect).

The compositions of the present invention can be administered by variousmethods known in the art. The particular mode selected will depend ofcourse, upon the particular composition selected, the severity of thecondition being treated and the dosage required for therapeuticefficacy. The methods of the invention, generally speaking, can bepracticed using any mode of administration that is medically acceptable,meaning any mode that produces effective levels of the active compoundswithout causing clinically unacceptable adverse effects. Such modes ofadministration include oral, rectal, parenteral, intracistemal,intravaginal, intraperitoneal, topical (as by powders, ointments, dropsor transdermal patch), bucal, or as an oral or nasal spray. The term“parenteral” as used herein refers to modes of administration whichinclude intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion. The compositionof the invention can also be injected directly in a lymph node.

Components of compositions for administration include sterile aqueous(e.g., physiological saline) or non-aqueous solutions and suspensions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Carriers or occlusive dressings can be used toincrease skin permeability and enhance antigen absorption.

Combinations can be administered either concomitantly, e.g., as anadmixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

Dosage levels depend on the mode of administration, the nature of thesubject, and the quality of the carrier/adjuvant formulation. Typicalamounts are in the range of about 0.1 μg to about 20 mg per subject.Preferred amounts are at least about 1 μg to about 1 mg, more preferablyat least about 10 to about 400 μg per subject. Multiple administrationto immunize the subject is preferred, and protocols are those standardin the art adapted to the subject in question.

The compositions can conveniently be presented in unit dosage form andcan be prepared by any of the methods well-known in the art of pharmacy.Methods include the step of bringing the compositions of the inventioninto association with a carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the compositions of the invention into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration can be presented asdiscrete units, such as capsules, tablets or lozenges, each containing apredetermined amount of the compositions of the invention. Othercompositions include suspensions in aqueous liquids or non-aqueousliquids such as a syrup, an elixir or an emulsion.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compositions of the invention described above,increasing convenience to the subject and the physician. Many types ofrelease delivery systems are available and known to those of ordinaryskill in the art.

Other embodiments of the invention include processes for the productionof the compositions of the invention and methods of medical treatmentfor cancer and allergies using said compositions.

Further aspects and embodiments of the present invention will becomeapparent in the following examples and the appended claims.

The following examples are illustrative only and are not intended tolimit the scope of the invention as defined by the appended claims. Itwill be apparent to those skilled in the art that various modificationsand variations can be made in the methods of the present inventionwithout departing from the spirit and scope of the invention. Thus, itis intended that the present invention cover the modifications andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

All patents and publications referred to herein are expresslyincorporated by reference in their entirety.

Example 1

Generation of p33-HBcAg VLPs.

The DNA sequence of HBcAg containing peptide p33 from LCMV is given inSEQ ID NO: 15. The p33-HBcAg VLPs were generated as follows: Hepatitis Bclone pEco63 containing the complete viral genome of Hepatitis B viruswas purchased from ATCC. The gene encoding HBcAg was introduced into theEcoRI/HindIII restriction sites of expression vector pkk223.3(Pharmacia) under the control of a strong tac promoter. The p33 peptide(KAVYNFATM) (SEQ ID NO: 82) derived from lymphocytic choriomeningitisvirus (LCMV) was fused to the C-terminus of HBcAg (1-185) via a threeleucine-linker by standard PCR methods. A clone of E. coli K802 selectedfor good expression was transfected with the plasmid, and cells weregrown and resuspended in 5 ml lysis buffer (10 mM Na2HPO4, 30 mM NaCl,10 mM EDTA, 0.25% Tween-20, pH 7.0). 200 μl of lysozyme solution (20mg/ml) was added. After sonication, 4 μl Benzonase and 10 mM MgCl2 wasadded and the suspension was incubation for 30 minutes at RT,centrifuged for 15 minutes at 15,000 rpm at 4° C. and the supernatantwas retained.

Next, 20% (w/v) (0.2 g/ml lysate) ammonium sulfate was added to thesupernatant. After incubation for 30 minutes on ice and centrifugationfor 15 minutes at 20,000 rpm at 4° C. the supernatant was discarded andthe pellet resuspended in 2-3 ml PBS. 20 ml of the PBS-solution wasloaded onto a Sephacryl 5-400 gel filtration column (Amersham PharmaciaBiotechnology AG), fractions were loaded onto a SDS-Page gel andfractions with purified p33-VLP capsids were pooled. Pooled fractionswere loaded onto a Hydroxyapatite column. Flow through (which containspurified p33-VLP capsids) was collected and loaded onto a reducingSDS-PAGE gel for monomer molecular weight analysis. Electron microscopywas performed according to standard protocols.

Thus, the structure of the p33-VLPs was assessed by electron microscopyand SDS PAGE. Recombinantly produced HBcAg wild-type VLPs (composed ofHBcAg [aa 1-185] monomers) and p33-VLPs were loaded onto a SephacrylS-400 gel filtration column (Amersham Pharmacia Biotechnology AG) forpurification. Pooled fractions were loaded onto a Hydroxyapatite column.Flow through (which contains purified p33-VLPs) was collected and loadedonto a reducing SDS-PAGE gel for monomer molecular weight analysis.

Throughout the description the terms p33-HBcAg VLP, HBcAg-p33 VLP,p33-VLPs and HBc33 are used interchangeably.

Example 2

Cloning, Expression and Purification of GA VLP

The cDNA of GA phage coat protein was amplified from GA phage by reversetranscription followed by a PCR amplification step, using the RevertAidFirst strand cDNA synthesis Kit (Fermentas). The cDNA was cut with theenzymes NcoI and HindIII, and cloned in vector pQβ185 previously cutwith the same enzymes, leading to plasmid 355.24, harboring GA cDNA. Thesequence of the inserted cDNA was checked by DNA sequencing.

Plasmid 355.24 was transformed in E. coli JM109. Expression wasperformed essentially as described for Qβ VLP. A single colony wasinoculated in LB medium containing 20 mg/L Ampicillin overnight withoutshaking. This inoculum was transferred the next day into a larger flaskcontaining M9 medium supplemented with 1% casaminoacids, 0.2% glucoseand 20 mg/L Ampicillin, and incubated under shaking for 14-20 h.

GA VLP was isolated essentially as described for Qβ VLP. Cells werelysed, and the cleared lysate was loaded onto a Sepharose CL-4B column(Amersham Pharmacia). The eluate was concentrated by ammonium sulphateprecipitation, and rechromatographed onto a Sepharose CL-6B column(Amersham Pharmacia). The final step was either an ultracentrifugationon sucrose gradient (20-50% w/v), or on CsCl. The isolated VLPs weresubsequently dialysed against 20 mM Tris, 150 mM NaCl, pH 8.0.

Example 3

Fluorescein Labeled CpG-Containing Oligonucleotides can be Packaged intoBKV VLPs.

VLPs produced in yeast contain small amounts of RNA which can be easilydigested and so eliminated by incubating the VLPs with RNase A. Thehighly active RNase A enzyme has a molecular weight of about 14 kDa andis small enough to enter the VLPs to eliminate the undesired ribonucleicacids. Recombinantly produced BKV VLPs (SEQ ID NO: 12) were concentratedto 1 mg/ml in PBS buffer pH7.2 and incubated in the absence or presenceof RNase A (200 μg/ml, Roche Diagnostics Ltd, Switzerland) for 3 h at37° C. After RNase A digestion BKV VLPs are supplemented with 75 nmol/ml5′-fluorescein labeled phosphorothioate G8-8-FAM oligonucleotide(oligonucleotide from SEQ ID NO: 7) and incubated for 3 h at 37° C.Subsequently BKV VLPs are subjected to DNaseI digestion for 3 h at 37°C. (40 u/ml AMPD1, Sigma, Division of Fluka AG, Switzerland) or loadedwithout DNaseI digestion. The samples were complemented with 6-foldconcentrated DNA-loading buffer (10 mM Tris pH7.5, 10% v/v glycerol,0.4% orange G) and run for 1 h at 65 volts in a 0.8% native tris-acetatepH 7.5 agarose gel.

BKV VLPs (15 μg) was analyzed by a native 0.8% agarose gelelectrophoresis after control incubation or after digestion with RNase Aand subsequent incubation with CpG-oligonucleotides (with phosphate- orwith phosphorothioate (pt) backbone) upon staining with ethidium bromideor Coomassie Blue.

Example 4

CpG-Containing Oligonucleotides can be Packaged into BKV VLPs.

To introduce immunostimulatory CpG-oligonucleotides, the RNase A treatedrecombinant BKV VLPs (Example 3) are supplemented with 150 nmol/ml G8-8oligonucleotides with phosphodiester backbone or G8-8 withphosphorothioate backbone and incubated for 3 h at 37° C. VLPpreparations for mouse immunization are extensively dialysed(10,000-fold diluted) for 24 h against PBS pH7.2 with a 300 kDa MWCOdialysis membrane (Spectrum Medical industries Inc., Houston, USA) toeliminate RNase A and the excess of CpG-oligonucleotides. The samplesare complemented with 6-fold concentrated DNA-loading buffer (10 mM TrispH7.5, 10% v/v glycerol, 0.4% orange G) and run for 1 h at 65 volts in a0.8% native tris-acetate pH7.5 agarose gel. BKV VLPs (15 μg) are loadedon a native 0.8% agarose gel electrophoresis and analyzed after controlincubation or after digestion with RNase A and subsequent incubationwith G8-8-oligonucleotides (with phosphodiester- or withphosphorothioate backbone) upon staining with ethidium bromide orCoomassie Blue in order to assess the presence of RNA/DNA or protein andthe reduction of unbound CpG-oligonucleotides after dialysis.

Example 5

Immunostimulatory Nucleic Acids can be Packaged into HBcAg VLPsComprising Fusion Proteins with Antigens.

HBcAg VLPs, when produced in E. coli by expressing the Hepatitis B coreantigen fusion protein p33-HBcAg (HBc33) (see Example 1) or the fusionprotein to the peptide P1A (HBcP1A), contain RNA which can be digestedand so eliminated by incubating the VLPs with RNase A.

The gene P1A codes for a protein that is expressed by the mastocytomatumor cell line P815. The dominant CTL epitope, termed P1A peptide,binds to MHC class I (Ld) and the complex is recognized by specific CTLclones (Brändle et al., 1998, Eur. J. Immunol. 28: 4010-4019). Fusion ofpeptide P1A-1 (LPYLGWLVF) ((SEQ ID NO: 95) to the C-terminus of HBcAg(aa 185, see Example 1) was performed by PCR using appropriate primersusing standard molecular biology techniques. A three leucine linker wascloned between the HBcAg and the peptide sequence. Expression wasperformed as described in Example 1. The fusion protein of HBcAg withP1A, termed HBcP1A, formed capsids when expressed in E. coli which couldbe purified similar to the procedure described in Example 1.

Enzymatic RNA hydrolysis: Recombinantly produced HBcAg-p33 (HBc33) andHBcAg-P1A (HBcP1A) VLPs at a concentration of 1.0 mg/ml in 1×PBS buffer(KCl 0.2 g/L, KH2PO4 0.2 g/L, NaCl 8 g/L, Na2HPO4 1.15 g/L) pH 7.4, wereincubated in the presence of 300 μg/ml RNase A (Qiagen AG, Switzerland)for 3 h at 37° C. in a thermomixer at 650 rpm.

Packaging of immunostimulatory nucleic acids: After RNA digestion withRNAse A HBcAg-p33 VLPs are supplemented with 130 nmol/mlCpG-oligonucleotides G3-6, G6 and G8-8 (Table 1). The resulting plasmid,produced in E. coli XL1-blue and isolated using the Qiagen Endofreeplasmid Giga Kit, is digested with restriction endonucleases XhoI andXbaI and resulting restriction products are separated by agaroseelectrophoresis. Inserts are isolated by electro-elution and ethanolprecipitation. Sequences are verified by sequencing of both strands.

DNAse I treatment: Packaged HBcAg-p33 VLPs are subsequently subjected toDNaseI digestion (5 U/ml) for 3 h at 37° C. (DNaseI, RNase free FlukaAG, Switzerland) and were extensively dialysed (2× against 200-foldvolume) for 24 h against PBS pH 7.4 with a 300 kDa MWCO dialysismembrane (Spectrum Medical industries Inc., Houston, USA) to eliminateRNAse A and the excess of CpG-oligonucleotides.

Benzonase treatment: Since some single stranded oligodeoxynucleotidesare partially resistant to DNaseI treatment, Benzonase treatment is usedto eliminate free oligonucleotides from the preparation. 100-120 U/mlBenzonase (Merck KGaA, Darmstadt, Germany) and 5 mM MgCl2 are added andincubated for 3 h at 37° C. before dialysis.

Dialysis: VLP preparations packaged with immunostimulatroy nucleic acidsused in mouse immunization experiments are extensively dialysed (2×against 200 fold volume) for 24 h against PBS pH 7.4 with a 300 kDa MWCOdialysis membrane (Spectrum Medical Industries, Houston, US) toeliminate added enzymes and free nucleic acids. Analytics of packaging:release of packaged immunostimulatory nucleic acids: To 50 μl capsidsolution 1 μl of proteinase K (600 U/ml, Roche, Mannheim, Germany), 3 μl10% SDS-solution and 6 μl 10 fold proteinase buffer (0.5 M NaCl, 50 mMEDTA, 0.1 M Tris pH 7.4) are added and subsequently incubated overnightat 37° C. VLPs are completed hydrolysed under these conditions.Proteinase K was inactivated by heating for 20 min at 65° C. 1 μl RNAseA (Qiagen, 100 μg/ml, diluted 250 fold) was added to 25 μl of capsid.2-30 μg of capsid were mixed with 1 volume of 2× loading buffer (1×TBE,42% w/v urea, 12% w/v Ficoll, 0.01% Bromphenolblue), heated for 3 min at95° C. and loaded on a 10% (for oligonucleotides of about 20 nt length)or 15% (for > than 40 mer nucleic acids) TBE/urea polyacrylamid gel(Invitrogen). Alternatively samples are loaded on a 1% agarose gel with6× loading dye (10 mM Tris pH 7.5, 50 mM EDTA, 10% v/v glycerol, 0.4%orange G). TBE/urea gels are stained with SYBRGold and agarose gels withstained with ethidium bromide.

Example 6

Immunostimulatory Nucleic Acids can be Packaged in HBcAg-wt Coupled withAntigens.

Recombinantly produced HBcAg-wt VLPs were packaged after coupling withpeptide p33 (CGG-KAVYNFATM) (SEQ ID NO: 83), derived from lymphocyticchoriomeningitis virus (LCMV). For coupling HBcAg-wt VLPs (2 mg/ml) werederivatized with 25× molar excess of SMPH(Succinimidyl-6-[(β-maleimido-propionamido)hexanoate], Pierce) for 1 hat 25° C. in a thermomixer. The derivatized VLPs were dialyzed to Mesbuffer (2-(N-morpholino) ethanesulphonic acid) pH 7.4 for 2×2 h usingMWCO 10.000 kD dialysis membranes at 4° C. VLPs (50 μM) weresubsequently coupled to the N-terminal cysteine of the p33 peptide (250μM) during a 2 h incubation in a thermomixer at 25° C. Samples weredialyzed (MWCO 300.000) extensively to 1×PBS pH 7.4 to eliminateundesired free peptide.

HBcAg-wt VLPs derivatization with SMPH and coupling to p33 peptide wasanalyzed on SDS-PAGE. Samples were analysed by 16% SDS PAGE and stainedwith Coomassie Blue. Loaded on the gel were the following samples: 1.NEB Prestained Protein Marker, Broad Range (#7708S), 10 μl; 2. p33peptide; 3. HBcAg-wt VLP derivatized with SMPH, before dialysis; 4.HBcAg-wt VLP derivatized with SMPH, after dialysis; 5. HBcAg-wt VLPcoupled with p33, supernatant; 6. HBcAg-wt VLP coupled with p33, pellet.HBcAg-wt was visible as a 21 kD protein band. Due to the low molecularweight of SMPH is the derivatised product only slightly larger and cannot be distinguished by SDS-PAGE. Peptide alone was visible as a 3 kDband and coupled product, termed HBx33, showed a strong secondary bandat approximately 24 kD accounting for more than 50% of total HBcAg-wt.

Enzymatic RNA hydrolysis: HBx33 VLPs (0.5-1.0 mg/ml, 1×PBS buffer pH7.4)in the presence of RNase A (300 μg/ml, Qiagen AG, Switzerland) werediluted with 4 volumes H2O to decrease salt concentration to a final0.2×PBS concentration and incubated for 3 h at 37° C. in a thermomixerat 650 rpm.

Packaging of immunostimulatory nucleic acids: After RNase A digestionHBx33 VLPs are concentrated using Millipore Microcon or Centriplusconcentrators, then supplemented with 130 nmol/ml G3-6, G6 or G8-8(Table 1) and incubated in a thermomixer for 3 h at 37° C. in 0.2×PBS pH7.4. Subsequently, reaction mixtures are subjected to DNaseI digestion(5 U/ml) for 3 h at 37° C. (DNaseI, RNase free Fluka AG, Switzerland).VLP preparations for mouse immunization were extensively dialysed (2×against 200-fold volume) for 24 h against PBS pH 7.4 with a 300 kDa MWCOdialysis membrane (Spectrum Medical industries Inc., Houston, USA) toeliminate RNase A and the excess of CpG-oligonucleotides.

Example 7

Immunostimulatory Nucleic Acids can be Packaged into Qβ VLPs Coupledwith Antigens.

Coupling of p33 Peptides to Qβ VLPs:

Recombinantly produced virus-like particles of the RNA-bacteriophage Qb(Qβ VLPs) were used untreated or after coupling to p33 peptidescontaining an N-terminal CGG or and C-terminal GGC extension(CGG-KAVYNFATM (SEQ ID NO: 83) and KAVYNFATM-GGC (SEQ ID NO: 84)).Recombinantly produced Qβ VLPs were derivatized with a 10 molar excessof SMPH (Pierce) for 0.5 h at 25° C., followed by dialysis against 20 mMHEPES, 150 mM NaCl, pH 7.2 at 4° C. to remove unreacted SMPH. Peptideswere added in a 5 fold molar excess and allowed to react for 2 h in athermomixer at 25° C. in the presence of 30% acetonitrile. The analysisof the p33 coupling to Qb VLPs was done on SDS-PAGE after Coomassie Bluestaining. Loaded were the following samples: (A) 1. NEB PrestainedProtein Marker, Broad Range (#7708S), 10 μl; 2. Qb VLP, 14 μg; 3. Qb VLPderivatized with SMPH, after dialysis; 4. Qb VLP coupled with CGG-p33,supernatant. (B) 1. NEB Prestained Protein Marker, Broad Range (#77085),10 μl; 2. Qb VLP, 10 μg; 3. Qb VLP coupled with GGC-p33, supernatant.The SDS-PAGE analysis demonstrated multiple coupling bands consisting ofone, two or three peptides coupled to the Qβ monomer. For the sake ofsimplicity the coupling product of the peptide p33 and Qβ VLPs wastermed, in particular, throughout the example section Qbx33.

Qβ VLPs, when produced in E. coli by expressing the bacteriophage Qβcapsid protein, contain RNA which can be digested and so eliminated byincubating the VLPs with RNase A.

Low Ionic Strength and Low Qβ Concentration Allow RNA Hydrolysis of QβVLPs by RNAse A:

Qβ VLPs at a concentration of 1.0 mg/ml in 20 mM Hepes/150 mM NaClbuffer (HBS) pH 7.4 were either digested directly by addition of RNase A(300 μg/ml, Qiagen AG, Switzerland) or were diluted with 4 volumes H2Oto a final 0.2×HBS concentration and then incubated with RNase A (60μg/ml, Qiagen AG, Switzerland). Incubation was allowed for 3 h at 37° C.in a thermomixer at 650 rpm. RNA hydrolysis from Qb VLPs by RNase Aunder low and high ionic strength was analyzed on a 1% agarose gelstained with ethidium bromide and Coomassie Blue. Loaded on the gel werethe following samples: (A, B) 1. MBI Fermentas 1 kb DNA ladder; 2. QbVLP untreated; 3. Qb VLP treated with RNase A in 1×HBS buffer pH7.2. (C,D) 1. MBI Fermentas 1 kb DNA ladder; 2. Qb VLP untreated; 3. Qb VLPtreated with RNase A in 0.2×HBS buffer pH7.2. It was demonstrated thatin 1×HBS only a very weak reduction of RNA content was observed (FIG.25A), while in 0.2×HBS most of the RNA were hydrolysed. In agreement,capsid migration was unchanged after addition of RNAse A in 1×HBS, whilemigration was slower after addition of RNAse in 0.2×HBS.

Low Ionic Strength Increases Nucleic Acid Packaging in Qβ VLPs:

After RNase A digestion in 0.2×HBS the Qβ VLPs are concentrated to 1mg/ml using Millipore Microcon or Centriplus concentrators and aliquotsare dialysed against 1×HBS or 0.2×HBS. Qβ VLPs are supplemented with 130nmol/ml G3-6, G6 or G8-8 (Table 1) and incubated in a thermomixer for 3h at 37° C. Subsequently Qβ VLPs are subjected to Benzonase digestion(100 U/ml) for 3 h at 37° C. Samples are analysed on 1% agarose gelsafter staining with ethidium bromide or Coomassie Blue.

Different Immunostimulatory Nucleic Acids can be Packaged in Qβ andQbx33 VLPs:

After RNase A digestion in 0.2×HBS the Qβ VLPs or Qbx33 VLPs areconcentrated to 1 mg/ml using Millipore Microcon or Centriplusconcentrators and supplemented with 130 nmol/ml G3-6, G6 and G8-8(Table 1) and incubated in a thermomixer for 3 h at 37° C. SubsequentlyQβ VLPs or Qbx33 VLPs are subjected to DNAse I digestion (5 U/ml) orBenzonase digestion (100 U/ml) for 3 h at 37° C. Samples are analysed on1% agarose gels after staining with ethidium bromide or Coomassie Blue.Packaging of G3-6, G6 or G8-8 can be analyzed by release of the nucleicacid by proteinase K digestion followed by agarose electrophoresis andethidium bromide staining.

Example 8

AP205 Disassembly-Purification-Reassembly and Packaging ofImmunostimulatory Nucleic Acids.

A. Disassembly and Reassembly of AP205 VLP from Material able toReassemble Without Addition of Oligonucleotide

Disassembly: 40 mg of lyophilized purified AP205 VLP (SEQ-ID: 80 or 81)were resolubilized in 4 ml 6 M GuHCl, and incubated overnight at 4° C.The disassembly mixture was centrifuged at 8000 rpm (Eppendorf 5810 R,in fixed angle rotor F34-6-38, used in all the following steps). Thepellet was resolubilized in 7 M urea, while the supernatant was dialyzed3 days against NET buffer (20 mM Tris-HCl, pH 7.8 with 5 mM EDTA and 150mM NaCl) with 3 changes of buffer. Alternatively, dialysis was conductedin continuous mode over 4 days. The dialyzed solution was centrifuged at8000 rpm for 20 minutes, and the pellet was resolubilized in 7 M urea,while the supernatant was pelletted with ammonium sulphate (60%saturation), and resolubilized in a 7 M urea buffer containing 10 mMDTT. The previous pellets all resolubilized in 7 M urea were joined, andprecipitated with ammonium sulphate (60% saturation), and resolubilizedin a 7 M urea buffer containing 10 mM DTT. The materials resolubilizedin the 7 M urea buffer containing 10 mM DTT were joined and loaded on aSephadex G75 column equilibrated and eluted with the 7 M urea buffercontaining 10 mM DTT at 2 ml/h, One peak eluted from the column.Fractions of 3 ml were collected. The peak fractions containing AP205coat protein were pooled and precipitated with ammonium sulphate (60%saturation). The pellet was isolated by centrifugation at 8000 rpm, for20 minutes. It was resolubilized in 7 M urea, 10 mM DTT, and loaded on ashort Sepharose 4B column (1.5×27 cm Sepharose 4B, 2 ml/h, 7 M urea, 10mM DTT as elution buffer). Mainly one peak, with a small shoulder elutedfrom the column. The fractions containing the AP205 coat protein wereidentified by SDS-PAGE, and pooled, excluding the shoulder. This yieldeda sample of 10.3 ml. The protein concentration was estimatedspectrophotometrically by measuring an aliquot of protein diluted25-fold for the measurement, using the following formula:(1.55×OD280−0.76×OD260)×volume. The average concentration was of 1nmol/ml of VLP (2.6 mg/ml). The ratio of absorbance at 280 nm vs. 260 nmwas of 0.12/0.105.

Reassembly: 1.1 ml beta-mercaptoethanol was added to the sample, and thefollowing reassembly reactions are set up:

1 ml of AP205 coat protein, no nucleic acids

1 ml of AP205 coat protein, rRNA (approx. 200 OD260 units, 10 nmol)

9 ml of AP205 coat protein, G8-8 (370 ul of 225 pmol/μl solution, i.e.83 nmol).

These mixtures are dialyzed 1 hour against 30 ml of NET buffercontaining 10% beta-mercaptoethanol. The mixture containing no nucleicacids is dialyzed separately. The dialysis is then pursued in acontinuous mode, and 1 l of NET buffer is exchanged over 3 days. Thereaction mixtures were subsequently extensively dialyzed against water(5 changes of buffer), and lyophilized. They are resolubilized in water,and analyzed by electron microscope (EM). All mixtures containedcapsids, showing that AP205 VLP reassembly is independent of thepresence of detectable nucleic acids, as measured by agarose gelelectrophoresis using ethidium bromide staining. The EM procedure is asfollows: A suspension of the proteins was absorbed on carbon-formvarcoated grids and stained with 2% phosphotungstic acid (pH 6,8). Thegrids were examined with a JEM 100C (JEOL, Japan) electron microscope atan accelerating voltage of 80 kV. Photographic records (negatives) areperformed on Kodak electron image film and electron micrographs wereobtained by printing of negatives on Kodak Polymax paper. The VLPreassembled in the presence of the G8-8 is purified over a Sepharose 4Bcolumn (1×50 cm), eluted with NET buffer (1 ml/h). The fractions areanalyzed by Ouchterlony assay, and the fractions containing VLP arepooled. Samples of the reassembly reaction containing G8-8 taken afterthe reassembly step and before extensive dialysis are analysed on a 0.6%agarose gel stained with ethidium-bromide and Coomassie blue.

B. Reassembly of AP205 VLP using Disassembled Material which does notReassemble in the Absence of Added Oligonucleotide

Disassembly: 100 mg of purified and dried recombinant AP205 VLP are usedfor disassembly as described above. All steps are performed essentiallyas described under disassembly in part A, but for the use of 8 M urea tosolublize the pellets of the ammonium sulphate precipitation steps andthe omission of the gel filtration step using a CL-4B column prior toreassembly. The pooled fractions of the Sephadex G-75 column contain 21mg of protein as determined by spectroscopy using the formula describedin part A. The ratio of absorbance at 280 nm to the absorbance at 260 nmof the sample is of 0.16 to 0.125. The sample is diluted 50 times forthe measurement.

Reassembly: The protein preparation resulting from the Sephadex G-75 gelfiltration purification step is precipitated with ammonium sulphate at60% saturation, and the resulting pellet solubilized in 2 ml 7 M urea,10 mM DTT. The sample is diluted with 8 ml of 10% 2-mercaptoethanol inNET buffer, and dialyzed for 1 hour against 40 ml of 10%2-mercaptoethanol in NET buffer. Reassembly is initiated by adding 0.4ml of a G8-8 solution (109 nmol/ml) to the protein sample in thedialysis bag. Dialysis in continuous mode is set up, and NET buffer usedas eluting buffer. Dialysis is pursued for two days and a sample istaken for EM analysis after completion of this dialysis step. Thedialyzed reassembly solution is subsequently dialyzed against 50% v/vGlycerol in NET buffer, to achieve concentration. One change of bufferis effected after one day of dialysis. The dialysis is pursued over atotal of three days.

The dialyzed and concentrated reassembly solution is purified by gelfiltration over a Sepharose 4-B column (1×60 cm) at a flow rate of 1ml/hour, in NET buffer. Fractions are tested in an Ouchterlony assay,and fractions containing capsids are dried, resuspended in water, andrechromatographed on the 4-B column equilibrated in 20 mM Hepes pH 7.6.Using each of the following three formula:

1. (183*OD230 nm−75.8*OD260 nm)*volume(ml) 2. ((OD235 nm−OD280nm)/2.51)×volume−3. ((OD228.5 nm−OD234.5 nm)*0.37)×volume proteinamounts of 6-26 mg of reassembled VLP were determined.

The reassembled AP205 VLPs are analyzed by EM as described above,agarose gel electrophoresis and SDS-PAGE under non-reducing conditions.

C. Coupling of p33 Epitope (Sequence: H2N-KAVYNFATMGGC-COON, with FreeN- and C-termini (SEQ ID NO: 54)) to AP205 VLPs Reassembled with G8-8

Reassembled AP205 VLP obtained as described in part B, and in 20 mMHepes, 150 mM NaCl, pH 7.4 is reacted at a concentration of 1.4 mg/mlwith a 5-fold excess of the crosslinker SMPH diluted from a 50 mM stockin DMSO for 30 minutes at 15° C. The obtained so-called derivatizedAP205 VLP is dialyzed 2×2 hours against at least a 1000-fold volume of20 mM Hepes, 150 mM NaCl, pH 7.4 buffer. The derivatized AP205 isreacted at a concentration of 1 mg/ml with either a 2.5-fold, or with a5-fold excess of Peptide, diluted from a 20 mM stock in DMSO, for 2hours at 15° C. The sample is subsequently flash frozen in liquidnitrogen for storage.

The coupling reaction is analyzed on an SDS-PAGE.

Example 9

Non-Enzymatic Hydrolysis of the RNA Content of VLPs and Packaging ofImmunostimulatory Nucleic Acids.

ZnSO4 Dependent Degradation of the Nucleic Acid Content of a VLP:

5 mg Qβ VLP (as determined by Bradford analysis) in 20 mM HEPES, pH 7.4,150 mM NaCl was dialysed either against 2000 ml of 50 mM TrisHCl pH 8.0,50 mM NaCl, 5% glycerol, 10 mM MgCl2 or 2000 ml of 4 mM HEPES, pH 7.4,30 mM NaCl for 2 h at 4° C. in SnakeSkin™ pleated dialysis tubing(Pierce, Cat. No. 68035). Each of the dialysis buffers was exchangedonce and dialysis was allowed to continue for another 16 h at 4° C. Thedialysed solution was clarified for 10 minutes at 14 000 rpm (Eppendorf5417 R, in fixed angle rotor F45-30-11, used in all the following steps)and protein concentration was again determined by Bradford analysis. QβVLPs in 50 mM TrisHCl pH 8.0, 50 mM NaCl, 5% glycerol, 10 mM MgCl2 werediluted with the corresponding buffer to a final protein concentrationof 1 mg/ml whereas Qβ VLPs in 4 mM HEPES pH 7.4, 30 mM NaCl were dilutedwith the corresponding buffer to a final protein concentration of 0.5mg/ml. This capsid-containing solutions were centrifuged again for 10minutes at 14 000 rpm at 4° C. The supernatants were than incubated withZnSO4 which was added to a final concentration of 2.5 mM for 24 h at 60°C. in an Eppendorf Thermomixer comfort at 550 rpm. After 24 h thesolutions were clarified for 10 minutes at 14000 rpm and the sedimentwas discarded. The efficiency of the ZnSO4-dependent degradation ofnucleic acids was confirmed by agarose gelelectrophoresis. Thesupernatants were dialysed against 5000 ml of 4 mM HEPES pH 7.4, 30 mMNaCl for 2 h at 4° C. 5000 ml buffer was exchanged once and dialysiscontinued over night at 4° C. The dialysed solution was clarified for 10minutes at 14 000 rpm and 4° C., a negligible sediment was discarded andthe protein concentration of the supernatants were determined byBradford analysis. Similar results were obtained with copperchloride/phenanthroline/hydrogen peroxide treatment of capsids. Thoseskilled in the art know alternative non-enzymatic procedures forhydrolysis or RNA.

ZnSO4-treated Qβ VLPs was analyzed by agarose gelelectrophoresis: QβVLPs which had been purified from E. coli and dialysed either againstbuffer 1 (50 mM TrisHCl pH 8.0, 50 mM NaCl, 5% glycerol, 10 mM MgCl2) orbuffer 2 (4 mM HEPES, pH 7.4, 30 mM NaCl) were incubated either withoutor in the presence of 2.5 mM zinc sulfate (ZnSO4) for 24 hrs at 60° C.After this treatment equal amounts of the indicated samples (5 μgprotein) were mixed with loading dye and loaded onto a 0.8% agarose gel.After the run the gel was stained with ethidium bromide. Treatment ofVLPs with ZnSO4 caused degradation of the nucleic acid content, whilethe mock-treated controls were unaffected.

Packaging of Oligodeoxynucleotides into ZnSO4-Treated VLPs:

ZnSO4-treated and dialysed Qβ capsids with a protein concentration (asdetermined by Bradford analysis) between 0.4 mg/ml and 0.9 mg/ml (whichcorresponds to a concentration of capsids of 159 nM and 357.5 nM,respectively) were used for the packaging of the oligodeoxynucleotides.The oligodeoxynucleotides were added at a 300-fold molar excess to theof Qβ-VLP capsids and incubated for 3 h at 37° C. in an EppendorfThermomixer comfort at 550 rpm. After 3 h the reactions were centrifugedfor 10 minutes at 14 000 rpm and 4° C. The supernatants were dialysed inSpectra/Por®CE DispoDialyzer with a MWCO 300′000 (Spectrum, Cat. No. 135526) against 5000 ml of 20 mM HEPES pH 7.4, 150 mM NaCl for 8 h at 4° C.5000 ml buffer was exchanged once and dialysis continued over night at4° C. The protein concentration of the dialysed samples were determinedby Bradford analysis. Qβ capsids and their nucleic acid contents wereanalyzed as described in Examples 5 and 7.

Packaging of oligodeoxynucleotides into ZnSO4-treated VLPs was analyzedby agarose gelelectrophoresis. Qβ VLPs which had been treated with 2.5mM zinc sulfate (+ZnSO4) were dialysed against 4 mM HEPES, pH 7.4, 30 mMNaCl and incubated for 3 hrs at 37° C. with an excess ofoligodeoxynucleotides (due to the dialysis the concentration of ZnSO4was decreased by an order of 106, therefore its indicated only inparenthesis) After this incubation in presence of oligodeoxynucleotides,equal amounts of the indicated samples (5 μg protein) were mixed withloading dye and loaded onto a 0.8% agarose gel. After the run the gelwas stained with ethidium bromide. Adding of oligodeoxynucleotides toZnSO4-treated Qβ VLPs could restore the electrophoretical behaviour ofthe so treated capsids when compared to untreated Qβ capsids which hadbeen purified from E. coli.

The nucleic acid content of ZnSO4- and oligodeoxynucleotide treated QβVLPs was analyzed by Benzonase and proteinase K digestion andpolyacrylamide TBE/Urea gelelectrophoresis: Oligodeoxynucleotides werepackaged into ZnSO4-treated Qβ VLPs as described above. 25 μg of theseVLPs were digested with 25 μl Benzonase (Merck, Cat. No. 1.01694.0001)according to the manufactures instructions. After heat-inactivation ofthe nuclease (30 minutes at 80° C.) the VLPs were treated withProteinase K (final enzyme concentration was 0.5 mg/ml) according to themanufactures instructions. After 3 hrs the equivalent of 2 ug Qβ VLPswhich had been digested by Benzonase and proteinase K were mixed withTBE-Urea sample buffer and loaded on a 15% polyacrylamide TBE-Urea gel(Novex®, Invitrogen Cat. No. EC6885). The capsids loaded in lane 2 weretreated with 2.5 mM ZnSO4 in presence of buffer 1 (see above), while thecapsids loaded in lane 3 were treated with 2.5 mM ZnSO4 in presence ofbuffer 2 (see above). As qualitative as well as quantitative standard, 1pmol, 5 pmol and 10 pmol of the oligodeoxynucleotide which was used forthe reassembly reaction, was loaded onto the same gel (lanes 4-6). Ascontrol, Qβ capsids which had been purified from E. coli were treatedexactly the same and analyzed on the same polyacrylamide TBE-Urea gel(lane 1). After the run was completed, the gel was fixed, equilibratedto neutral pH and stained with SYBR-Gold (Molecular Probes Cat. No.S-11494). Intact Qβ VLPs (which had been purified from E. coli) did notcontain nucleic acids of similar size than those which had beenextracted from ZnSO4- and oligodeoxynucleotide treated Qβ capsids. Inaddition, nucleic acids isolated from the latter VLPs were emigratingwith the oligodeoxynucleotides which had been used in the reassemblyreaction. These results confirmed that the used oligodeoxynucleotideswere packaged into ZnSO4-treated Qβ capsids.

Example 10

VLPs Containing Containing Immunostimulatory Nucleic Acids Induce T CellResponses that can be Boosted by Viral Vectors: LCMV.

Mice were subcutaneously primed with 20 μg p33-VLPs (see EXAMPLE 1)containing immunostimulatory nucleic acids. Before immunization, p33-VLPpreparations were extensively purified from unbound CpG-oligonucleotidesvia dialysis. 12 days later, blood was taken and frequencies ofp33-specific T cells were determined by tetramer staining. The mice wereboosted with 200 pfu of live LCMV strain WE and frequencies of specificT cells were determined 5 days later. Frequencies before boost were3.5%±1.8% and after boost 15.5%±1.9%.

Example 11

VLPs Containing Immunostimulatory Nucleic Acids Induce T Cell Responsesthat can be Boosted by Viral Vectors: Recombinant Vaccinia Virus.

Mice are subcutaneously primed with 20 μg p33-VLPs (see EXAMPLE 1)containing immunostimulatory nucleic acids. Before immunization, p33-VLPpreparations are extensively purified from unbound CpG-oligonucleotidesvia dialysis. 12 days later, blood is taken and frequencies ofp33-specific T cells are determined by tetramer staining. The mice areboosted with 106 pfu of recombinant vaccina virus expressing LCMV-GP andfrequencies of specific T cells are determined 5 days later.

Example 12

VLPs Containing Immunostimulatory Nucleic Acids Induce T Cell Responsesthat can be Boosted by Viral Vectors: Recombinant Canary Pox Virus.

Mice are subcutaneously primed with 20 μg p33-VLPs containingimmunostimulatory nucleic acids. Before immunization, p33-VLPpreparations are extensively purified from unbound CpG-oligonucleotidesvia dialysis. 12 days later, blood is taken and frequencies ofp33-specific T cells are determined by tetramer staining. The mice areboosted with 107 pfu of recombinant canary pox virus expressing LCMV-GPand frequencies of specific T cells are determined 5 days later.

Example 13

VLPs Containing Containing Immunostimulatory Nucleic Acids can Boost TCell Responses.

Mice are infected intravenously with recombinant vacccina virusexpressing LCMV-GP. 20 days later, blood is taken and frequencies ofp33-specific T cells are determined by tetramer staining. The mice areboosted the same day with p33-VLP preparations containingimmunostimulatory nucleic acids and frequencies of specific T cells aredetermined 5 days later.

Example 14

Coupling of Antigenic Peptides after Packaging of ImmunostimulatoryNucleic Acids into VLPs.

RNaseA and ZnSO4 mediated degradation of the nucleic acid content of aVLP.

Qβ VLPs were treated with RNaseA as described in Example 7 under lowionic strength conditions (20 mM Hepes pH 7.4 or 4 mM Hepes, 30 mM NaCl,pH 7.4). Similarly, other VLPs such as described in Examples 2, 3, 5,and 8, i.e. GA, BKV, HBcAg, and AP205 are treated. Alternatively, QβVLPs and AP205 VLPs were treated with ZnSO4 under low ionic strengthconditions (20 mM Hepes pH 7.4 or 4 mM Hepes, 30 mM NaCl pH 7.4) asdescribed in Example 9. AP205 VLP (1 mg/ml) in either 20 mM Hepes pH 7.4or 20 mM Hepes, 1 mM Tris, pH 7.4 was treated for 48 h with 2.5 mM ZnSO4at 50° C. in an Eppendorf Thermomixer comfort at 550 rpm. Qβ and AP205VLP samples were clarified as described in Example 9 and supernatantswere dialysed in 10.000 MWCO Spectra/Por® dialysis tubing (Spectrum,Cat. nr. 128 118) against first 21 20 mM Hepes, pH 7.4 for 2 h at 4° C.and, after buffer exchange, overnight. Samples were clarified afterdialysis as described in Example 9 and protein concentration in thesupernatants was determined by Bradford analysis.

Packaging of ISS into RnaseA and ZnSO4 Treated VLPs.

After RNA hydrolysis and dialysis, Qβ and AP205 VLPs (1-1.5 mg/ml) weremixed with 130 μl of CpG oligonucleotides (G3-6, G8-8—cf. Table 1; 1 mMoligonucleotide stock in 10 mM Tris pH 8) per ml of VLPs. Samples wereincubated for 3 h at 37° C. in a thermoshaker at 650 rpm. Subsequently,samples were treated with 125 U Benzonase/ml VLPs (Merck KGaA,Darmstadt, Germany) in the presence of 2 mM MgCl2 and incubated for 3 hat 37° C. before dialysis. Samples were dialysed in 300.000 MWCOSpectra/Por® dialysis tubing (Spectrum, Cat. nr. 131 447) against 20 mMHepes, pH 7.4 for 2 h at 4° C., and after buffer exchange overnightagainst the same buffer. After dialysis samples were clarified asdescribed in Example 9 and protein concentration in the supernatantswere determined by Bradford analysis.

Coupling of Immunogenic Peptides to ISS Packaged VLPs.

Qβ VLPs, packaged with ISS were coupled to p33 peptides containing aC-terminal GGC extension (KAVYNFATM-GGC) (SEQ ID NO: 84), resulting inQb VLPs termed Qb-ISS-33 VLPs. Packaged Qβ VLPs in 20 mM Hepes, pH 7.4were derivatized with a 10-fold molar excess of SMPH (Pierce) for 0.5 hat 25° C., followed by two dialysis steps of 2 hours each against 20 mMHEPES pH 7.4 at 4° C. to remove unreacted SMPH. Peptides were added in a5-fold molar excess to the dialysed derivatization mixture, and allowedto react for 2 h in a thermomixer at 25° C. Samples were dialysed in300.000 MWCO Spectra/Por® dialysis tubing against 20 mM Hepes pH 7.4 for2 h at 4° C., and after buffer exchange overnight against the samebuffer. After dialysis samples were clarified as described in Example 9and protein concentration in the supernatants were determined byBradford analysis. Coupling of peptide p33 to Qβ was analysed bySDS-PAGE on 16% PAGE Tris-Glycine gels (Novex® by Invitrogen, Cat. No.EC64952), using a sample buffer containing 2% SDS and β-mercapto ethanolor DTT. Packaging was analysed on 1% agarose gels and, after proteinaseK digestion, on TBE/urea gels as described in Example 5.

AP205 VLPs (1.24 mg/ml) packaged with G8-8 oligonucleotide as describedabove were derivatized and coupled to HIVp17 (71-85) containing aN-terminal GGC extension (CGG-GSEEIRSLYNTVATL) (SEQ ID NO: 85),resulting in AP205-G8-8-HIVp17 VLPs. AP205 VLPs (packaged with G8-8), in20 mM Hepes pH 7.4, were derivatized with a 20-fold molar excess of SMPHfor 0.5 h at 25° C., and subsequently dialysed two times against 20 mMHEPES, pH 7.4 at 4° C. to remove unreacted SMPH. Peptide was added tothe dialyzed derivatization mixture in a 10-fold molar excess andallowed to react for 2 h in a thermomixer at 25° C. Samples weredialysed in 10.000 MWCO dialysis tubing against 20 mM Hepes pH 7.4 for 2h at 4° C., and after buffer exchange, overnight against the samebuffer. After dialysis, samples were clarified as described in Example 9and protein concentration in the supernatants were determined byBradford analysis. Coupling efficiency of peptide HIVp17 to AP205 wasanalysed by SDS-PAGE on 16% PAGE Tris-Glycine gels. G8-8 oligonucleotidepackaging in AP205 was analysed on 1% agarose gels and, after proteinaseK digestion, G8-8 oligonucleotide amount in AP205-G8-8-HIVp17 wasanalysed on TBE/urea gels as described in Example 5.

Packaging of G8-8 oligonucleotides into Qβ VLPs and subsequent couplingto p33 peptide was analyzed by agarose gelelectrophoresis. Qβ VLPscontaining G8-8 oligonucleotides and subsequently coupled to p33 peptidewere termed Qb-G8-8-33 VLPs. Ethidium bromide staining of G8-8 packagedQβ VLPs can be seen on a 1% agarose gel stained with ethidium bromide.Comigration of the ethidium bromide fluorescent band with the Qβ VLPprotein band visible on the same gel subsequently stained with CoomassieBlue demonstrates packaging. Coupling efficiency can be estimated to be30% by SDS-PAGE analysis on a 16% PAGE Tris-Glycine gel. Analysis of theG8-8 content of Qb-G8-8-33 VLPs after coupling was done on a 1% agarosegel, where the amount of oligonucleotide packaged was of approximately 1nmol/100 μg Qb-G8-8-33 VLPs.

Packaging of G8-8 oligonucleotides into AP205 VLPs was analyzed bygelelectrophoresis. Staining of G8-8 packaged AP205 VLPs can be seen ona 1% agarose gel stained with ethidium bromide. Comigration of the AP205VLPs protein band detected on the same gel subsequently stained withCoomassie Blue demonstrated packaging. Coupling efficiency with theHIVp17 peptide could be estimated from the SDS-PAGE analysis on a 16%PAGE Tris-Glycine gel where multiple coupling bands migrating slowerthan the residual AP205 VLP monomer subunits, which did not react withpeptide, are visible. Coupling efficiency was comparable to the couplingefficiency obtained for the Qb-G8-8-33 VLPs. Analysis of the G8-8oligonucleotide content of AP205 VLPs after coupling to HIVp17 can beseen on TBE/urea gel electrophoresis indicating a packaged amount of0.5-1 nmol/100 μg AP205-G8-8-HIVp17 VLPs.

Example 15

Packaging of Immunostimulatory Guanosine Flanked Oligonucleotides intoVLPs.

Qbx33 VLPs (Qβ VLPs coupled to peptide p33, see Example 7) were treatedwith RNaseA under low ionic conditions (20 mM Hepes pH 7.4) as describedin Example 7 to hydrolyse RNA content of the Qbx33 VLP. After dialysisagainst 20 mM Hepes pH 7.4, Qbx33 VLPs were mixed with guanosine flankedoligonucleotides (Table 1: G3-6, G7-7, G8-8, G9-9 or G6, from a 1 mMoligonucleotide stock in 10 mM Tris pH 8) and incubated as described inExample 14. Subsequently, Qbx33 VLPs were treated with Benzonase anddialysed in 300.000 MWCO tubing. Samples with oligos G7-7, G8-8 and G9-9were extensively dialysed over 3 days with 4 buffer exchanges to removefree oligo. Packaging was analysed on 1% agarose gels and, afterproteinase K digestion, on TBE/urea gels as described in Example 5.

TABLE 1 Sequences of immunostimulatory nucleic acids used in theExamples. ISS name 5′-3′sequence SEQ ID NO GACGATCGTC 1 G3-6GGGGACGATCGTCGGGGGG 2 G4-6 GGGGGACGATCGTCGGGGGG 3 G5-6GGGGGGACGATCGTCGGGGGG 4 G6-6 GGGGGGGACGATCGTCGGGGGG 5 G7-7GGGGGGGGACGATCGTCGGGGGGG 6 G8-8 GGGGGGGGGACGATCGTCGGGGGGGG 7 G9-9GGGGGGGGGGACGATCGTCGGGGGGGGG 8 G6 GGGGGGCGACGACGATCGTCGTCGGGGGGG 9 Smallletters indicate deoxynucleotides connected via phosphorothioate bondswhile larger letters indicate deoxynucleotides connected viaphosphodiester bonds

Packaging of G3-6, G6 and G8-8 oligonucleotides in RNaseA treated Qbx33VLPs was analyzed by agarose gelelectrophoresis. Upon oligonucleotidepackaging, a fluorescent band migrating slightly slower than referenceuntreated Qβ VLP becomes visible on the 1% agarose gel stained withethidium bromide indicating the presence of oligonucleotides. The signalis maintained after treatment with Benzonase, indicating packaging ofoligonucleotides within the Qbx33 VLPs. The packaging efficiency can beestimated from the TBE/urea gel electrophoresis. The amount of the G3-6oligonucleotide (approximately 4 nmol/100 μg Qbx33 VLPs) packaged ismuch higher than the amount of packaged G8-8 oligonucleotide(approximately 1 nmol/100 μg Qbx33 VLPs). This indicates a dependence ofpackaging ability on the length of the guanosine nucleotides tailflanking the CpG motif.

Example 16

Packaging Ribonucleic Acid into VLPs.

ZnSO4 Dependent Degradation of the Nucleic Acid Content of a VLP.

Qβ VLPs were treated with ZnSO4 under low ionic strength conditions (20mM Hepes pH 7.4 or 4 mM Hepes, 30 mM NaCl, pH 7.4) as described inExample 9. AP205 VLPs (1 mg/ml) in either 20 mM Hepes pH 7.4 or 20 mMHepes, 1 mM Tris, pH 7.4 were treated for 48 h with 2.5 mM ZnSO4 at 50°C. in an Eppendorf Thermomixer comfort at 550 rpm. Qβ and AP205 VLPsamples were clarified as in Example 9 and dialysed against 20 mM Hepes,pH 7.4 as in Example 14.

Packaging of poly (I:C) into ZnSO4-Treated VLPs:

The immunostimulatory ribonucleic acid poly (I:C), (Cat. nr. 27-4732-01,poly(I)•poly(C), Pharmacia Biotech) was dissolved in PBS (Invitrogencat. nr. 14040) or water to a concentration of 4 mg/ml (9 μM). Poly(I:C) was incubated for 10 minutes at 60° C. and then cooled to 37° C.,Incubated poly (I:C) was added in a 10-fold molar excess to eitherZnSO4-treated Qβ or AP205 VLPs (1-1.5 mg/ml) and the mixtures wereincubated for 3 h at 37° C. in a thermomixer at 650 rpm. Subsequently,excess of free poly (I:C) was enzymatically hydrolysed by incubationwith 125 U Benzonase per ml VLP mixture in the presence of 2 mM MgCl2for 3 h at 37° C. in a thermomixer at 300 rpm. Upon Benzonase hydrolysissamples were clarified as described in Example 9 and supernatants weredialysed in 300.000 MWCO Spectra/Por® dialysis tubing (Spectrum, Cat.nr. 131 447) against 2120 mM Hepes, pH 7.4 for 2 h at 4° C., and afterbuffer exchange overnight against the same buffer. After dialysis,samples were clarified as described in Example 9 and proteinconcentration in the supernatants were determined by Bradford analysis.

Coupling of Immunogenic Peptides to poly (I:C) Packaged VLPs.

Qβ VLPs (1 mg/mi.) packaged with poly (I:C) were derivatized and coupledeither to p33 peptide (KAVYNFATM-GGC) (SEQ ID NO: 84) as described inExample 14, or to MelanA peptide (MelanA 16-35A/L CGHGHSYTTAEELAGIGILTV)(SEQ ID NO: 40), resulting in Qb-pIC-33 and Qb-pIC-MelanA VLPs,respectively. For coupling to MelanA peptide, the packaged Qβ VLP wasderivatized with a 2.1-fold molar excess of SMPH (Pierce) for 0.5 h at25° C., followed by two dialysis steps against 20 mM HEPES, pH 7.4 at 4°C. to remove unreacted SMPH. Peptides were added in a 2.1-fold molarexcess and allowed to react for 1.5 h in a thermomixer at 25° C. Sampleswere dialysed in 300.000 MWCO Spectra/Por® CE Dispo Dialyzer against 20mM Hepes, pH 7.2 for 3 h at 4° C., and after buffer exchange, overnightagainst the same buffer. After dialysis samples were clarified asdescribed in Example 9 and protein concentration in the supernatantswere determined by Bradford analysis. Coupling of peptide p33 andpeptide MelanA to Qβ was analysed by SDS-PAGE on 16% PAGE Tris-Glycinegels. Packaging was analysed on 1% agarose gels and, after proteinase Kdigestion, on TBE/urea gels as described in Example 5.

AP205 VLPs (1 mg/ml) packaged with poly (I:C) were derivatized andcoupled to HIVp17 (71-85) containing a N-terminal GGC extension(CGG-GSEEIRSLYNTVATL) (SEQ ID NO: 85), resulting in AP205-pIC-HIVp17VLPs. AP205 VLPs, in 20 mM Hepes, pH 7.4 were derivatized with a 20-foldmolar excess of SMPH for 0.5 h at 25° C., and subsequently dialysed twotimes against 20 mM HEPES, pH 7.4 at 4° C. to remove unreacted SMPH.Peptide was added to the dialyzed derivatization mixture in a 10-foldmolar excess and allowed to react for 2 h in a thermomixer at 25° C.Samples were dialysed in 10.000 MWCO dialysis tubing against 20 mM HepespH 7.4 for 2 h at 4° C., and after buffer exchange, overnight againstthe same buffer. After dialysis, samples were clarified as described inExample 9 and protein concentration in the supernatants were determinedby Bradford analysis. Coupling efficiency of peptide HIVp17 to AP205 wasanalysed by SDS-PAGE on 16% PAGE Tris-Glycine gels. Poly (I:C) packagingwas analysed on 1% agarose gels and, after proteinase K digestion, onTBE gels as described in Example 5.

Packaging of poly (I:C) into ZnSO4 treated Qβ VLPs and coupling withMelanA peptide resulting in Qb-pIC-MelanA VLPs was analyzed by agarosegelelectrophoresis. The fluorescent signal visible on an ethidiumbromide stained 1% agarose gel, indicating presence of nucleic acid,co-migrates with the protein band that became visible upon CoomassieBlue staining of the gel, demonstrating packaging. Coupling efficiencyof the MelanA peptide was estimated by SDS-PAGE analysis on a 16% PAGETris-Glycine gel. Multiple coupling products were visible as bandsmigrating slower than the Qβ VLP monomer subunits, which had not reactedwith peptide. Coupling efficiency of MelanA was overall comparable tothe coupling efficiency obtained for the Qb-G8-8-33 VLPs and theAP205-G8-8-HIVp17 VLPS of Example 14, albeit slightly lower. Thepackaging efficiency into Qb-pIC-MelanA could be estimated from theTBE/urea gel; the packaged amount of poly (I:C) in Qβ was approximately25 pmol and remained the same upon MelanA coupling.

Packaging of poly (I:C) into ZnSO4 treated AP205 VLPs and in thecoupling product AP205-pIC-HIVp17 after coupling to HIVp17 was analyzedby agarose gelelectrophoresis. The fluorescent band visible on anethidium bromide stained 1% agarose gel, indicating presence of nucleicacid, co-migrates with the protein band that became visible uponCoomassie Blue staining of the gel both before and after coupling toHIVp17. Coupling efficiency of the HIVp17 peptide is estimated from theappearance of multiple coupling products visible as bands migratingslower than AP205 VLP subunit monomer, which did not react with peptide,after. SDS-PAGE analysis on a 16% PAGE Tris-Glycine gel electrophoresis.Coupling efficiency was overall comparable to the coupling efficiencyobtained for the Qb-G8-8-33 VLPs and the AP205-G8-8-HIVp17 VLPs (Example14). The packaging efficiency could be estimated from the TBE gel, whichshowed that the packaged amounts of poly (I:C) in the AP205-pIC-HIVp17VLP is approximately 10 pmol/100 μg VLP.

Packaging of G8-8 into ZnSO4-treated VLPs and coupling of immunogenicpeptides to G8-8 packaged VLP can be performed accordingly.

Example 17

Packaging of Immunostimulatory Guanosine Flanked Oligonucleotides intoHBcAg VLPs.

HBcAg VLPs are treated with RNaseA under low ionic strength conditions(20 mM Hepes pH 7.4) as described in Example 7 to hydrolyse RNA contentof the VLP. After dialysis against 20 mM Hepes, pH 7.4, VLPs are mixedwith guanosine flanked oligonucleotides (Table 1; G3-6, G7-7, G8-8,G9-9, or G6, 1 mM stock in 10 mM Tris pH 8) and incubated as describedin Example 14. Subsequently, VLPs are treated with Benzonase anddialysed in 300.000 MWCO tubing. Packaging is analysed on 1% agarosegels and on TBE/urea gels after proteinase K digestion as described inExample 5.

Example 18

Packaging of Immunostimulatory Guanosine Flanked Oligonucleotides intoGA VLPs.

GA VLPs are treated with RNaseA under low ionic conditions (20 mM HepespH 7.4) as described in Example 7 to hydrolyse RNA content of the VLP.After dialysis against 20 mM Hepes pH 7.4, VLPs are mixed with guanosineflanked oligonucleotides (Table 1; G3-6, G7-7, G8-8, G9-9, or G6, 1 mMstock in 10 mM Tris pH8) and incubated as described in Example 14.Subsequently, VLPs are treated with Benzonase and dialysed in 300.000MWCO tubing. Packaging is analysed on 1% agarose gels and on TBE/ureagels after proteinase K digestion as described in Example 5.

Example 19

Packaging Ribonucleic Acid into HBcAg VLPs.

HBcAg VLPs are treated with ZnSO4 under low ionic strength conditions(20 mM Hepes pH 7.4 or 4 mM Hepes, 30 mM NaCl, pH 7.4) as described inExample 9 and are dialysed against 20 mM Hepes pH 7.4 as in Example 14.G8-8 is added in a 10-fold molar excess to HBcAg VLPs (1-1.5 mg/ml) andincubated for 3 h at 37° C. in a thermomixer at 650 rpm as described inExample 16. Subsequently, excess of free G8-8 is enzymaticallyhydrolysed by incubation with 125 U Benzonase per ml VLP mixture in thepresence of 2 mM MgCl2 for 3 h at 37° C. in a thermomixer at 300 rpm.Samples are clarified after Benzonase hydrolysis as described in Example9 and dialysed as in Example 16. After dialysis, samples are clarifiedas described in Example 9 and protein concentration in the supernatantsare determined by Bradford analysis. HBcAg VLPs (1 mg/ml) packaged withG8-8 are derivatized and coupled either to MelanA or to HIVp17 peptide,and dialysed as in Example 16.

Example 20

Packaging Ribonucleic Acid into GA VLPs.

GA VLPs are treated with ZnSO4 under low ionic strength conditions (20mM Hepes pH 7.4 or 4 mM Hepes, 30 mM NaCl, pH 7.4) as described inExample 9 and are dialysed against 20 mM Hepes, pH 7.4 as in Example 14.G8-8 is added in a 10-fold molecular excess to GA VLPs (1-1.5 mg/ml) andincubated for 3 h at 37° C. in a thermomixer at 650 rpm as described inExample 16. Subsequently, excess of free G8-8 is enzymaticallyhydrolysed by incubation with 125 U Benzonase per ml VLP mixture in thepresence of 2 mM MgCl2 for 3 h at 37° C. in a thermomixer at 300 rpm.Samples are clarified after Benzonase hydrolysis as described in Example9 and dialysed as in Example 16. After dialysis, samples are clarifiedas described in Example 9 and protein concentration in the supernatantsare determined by Bradford analysis. GA VLPs (1 mg/ml) packaged withG8-8 are derivatized and coupled either to MelanA or to HIVp17 peptide,and dialysed as in Example 16.

Example 21

Qβ Disassembly, Reassembly and Packaging of Oligodeoxynucleotides.

Disassembly and Reassembly of Qβ VLP

Disassembly: 45 mg Qβ VLP (2.5 mg/ml, as determined by Bradfordanalysis) in PBS (20 mM Phosphate, 150 mM NaCl, pH 7.5), was reducedwith 10 mM DTT for 15 min at RT under stirring conditions. A secondincubation of 15 min at RT under stirring conditions followed afteraddition of magnesium chloride to a final concentration of 700 mM,leading to precipitation of the encapsulated host cell RNA andconcomitant disintegration of the VLPs. The solution was centrifuged 10min at 4000 rpm at 4 ° C. (Eppendorf 5810 R, in fixed angle rotor A-4-62used in all following steps) in order to remove the precipitated RNAfrom the solution. The supernatant, containing the released, dimeric Qβcoat protein, was used for the chromatography purification steps.

Two-step purification method for Qβ coat protein by cation exchangechromatography and size exclusion chromatography: The supernatant of thedisassembly reaction, containing dimeric coat protein, host cellproteins and residual host cell RNA, was applied onto a SP-Sepharose FFcolumn (xk16/20, 6 ml, Amersham Bioscience). During the run, which wascarried out at RT with a flow rate of 5 ml/min, the absorbance at 260 nmand 280 nm was monitored. The column was equilibrated with 20 mM sodiumphosphate buffer pH 7 and the sample was diluted 1:15 in water to adjusta conductivity below 10 mS/cm in order to achieve proper binding of thecoat protein to the column. The elution of the bound coat protein wasaccomplished by a step gradient to 20 mM sodium phosphate/500 mM sodiumchloride and the protein was collected in a fraction volume of approx.25 ml. The column was regenerated with 0.5 M NaOH.

In the second step, the isolated Qβ coat protein dimer (the elutedfraction from the cation exchange column) was applied (in two rims) ontoa Sephacryl S-100 HR column (xk26/60, 320 ml, Amersham Bioscience)equilibrated with 20 mM sodium phosphate/250 mM sodium chloride; pH 6.5.Chromatography was performed at RT with a flow rate of 2.5 ml/min.Absorbance was monitored at 260 nm and 280 nm. Fractions of 5 ml werecollected. The column was regenerated with 0.5 M NaOH.

Reassembly by dialysis: A stock solution of purified Qβ coat proteindimer at a concentration of 2 mg/ml was used for the reassembly of QβVLP in the presence of the oligodeoxynucleotide G8-8. The concentrationof oligodeoxynucleotide in the reassembly mixture was 10 μM. Theconcentration of coat protein dimer in the reassembly mixture was 40 μM(approx. 1.13 mg/ml). Stock solutions of urea and DTT were added to thesolution to give final concentrations of 1 M urea and 5 mM DTTrespectively. The oligodeoxynucleotide was added as last component,together with H₂O, giving a final volume of the reassembly reaction of 3ml. This solution was dialysed at 4° C. for 72 h against 1500 ml buffercontaining 20 mM TrisHCl, 150 mM NaCl, pH 8.0. The dialysed reassemblymixture was centrifuged at 14 000 rpm for 10 minutes at 4° C. Anegligible sediment was discarded while the supernatant contained thereassembled and packaged VLPs. Reassembled and packaged VLPs wereconcentrated with centrifugal filter devices (Millipore, UFV4BCC25, 5KNMWL) to a final protein concentration of 3 mg/ml. Protein concentrationwas determined by Bradford analysis.

Purification of reassembled and packaged VLPs by size exclusionchromatography: Up to 10 mg total protein was loaded onto a Sepharose™CL-4B column (xk16/70, Amersham Biosciences) equilibrated with 20 mMHEPES, 150 mM NaCl, pH 7.4. The chromatography was performed at roomtemperature at a flow-rate of 0.4 ml/min. Absorbance was monitored at260 nm and 280 nm. Two peaks were observed, collected in fractions of0.5 ml size and analysed by SDS-PAGE. The disulfide-bond pattern inreassembled and purified Qβ capsids was analyzed by non-reducingSDS-PAGE. 5 μg of the indicated capsids were mixed with sample buffer(containing SDS) that contained no reducing agent and loaded onto a 16%Tris-Glycine gel. After the run was completed the gel was stained withCoomassie blue. When compared to “intact” capsids purified from E. coli,the reassembled Qβ VLP displayed the same disulfide bond pattern withthe bands corresponding to dimer, timer, tetramer, pentamer and hexamersof the Qb coat protein. Calibration of the column with intact and highlypurified Qβ capsids from E. coli, revealed that the apparent molecularweight of the major first peak was consistent with Qβ capsids.

Reassembly by diafiltration (optimized method): 20 ml of a stocksolution of purified coat protein (1.5 mg/ml) is mixed with stocksolutions of urea, DTT, oligodeoxynucleotide G8-8 and water. Theoligodeoxynucleotide is added as last component. The volume of themixture is 30 ml and the final concentrations of the components are 35μM dimeric coat protein (reflecting 1 mg/ml), 35 μMoligodeoxynucleotide, 1 M urea and 2.5 mM DTT. The mixture is thendiafiltrated against 300 ml of 20 mM sodium phosphate/250 mM sodiumchloride, pH 7.2, in a tangential flow filtration apparatus at RT, usinga Pellicon XL membrane cartridge (Biomax 5K, Millipore). The total flowrate is set to 10 ml/min and the permeate flow rate set to 2.5 ml/min.After completion of the diafiltration step, H₂O₂ is added to thesolution to a final concentration of 7 mM and the solution is furtherincubated at RT for 60 min, to accelerate the formation of thestructural disulfide bonds in the formed VLPs. The removal ofnon-incorporated oligodeoxynucleotide and coat protein is achieved by a2^(nd) diafiltration against 600 ml of 20 mM sodium phosphate/250 mMsodium chloride, pH 7.2, using a Pellicon XL membrane cartridge (PLCMK300K, Millipore).

Analysis of Qβ VLPs which had been Reassembled in the Presence ofOligodeoxynucleotides:

A) Hydrodynamic size of reassembled capsids: Qβ capsids, which had beenreassembled in the presence of oligodeoxynucleotide G8-8, were analyzedby dynamic light scattering (DLS) and compared to intact Qβ VLPs, whichhad been purified from E. coli. Reassembled capsids showed the samehydrodynamic size (which depends both on mass and conformation) as theintact Qβ VLPs.

B) Disulfide-bond formation in reassembled capsids: Reassembled Qβ VLPswere analyzed by non-reducing SDS-PAGE and compared to intact Qβ VLPs,which had been purified from E. coli. Reassembled capsids displayed aband pattern, with the presence of disulfide-linked pentameric andhexameric forms of the coat protein, similar to the intact Qβ VLPs (asdescribed above).

C) Analysis of nucleic acid content of the Qβ VLPs which had beenreassembled in the presence of oligodeoxynucleotides by denaturingpolyacrylamide TBE-Urea gelelectrophoresis: Reassembled Qβ VLPs (0.4mg/ml) containing G8-8 oligodeoxynucleotides were incubated for 2 h at37° C. with 125 U benzonase per ml Qβ VLPs in the presence of 2 mMMgCl₂. Subsequently the benzonase treated Qβ VLPs were treated withproteinase K (PCR-grade, Roche Molecular Biochemicals, Cat. No. 1964364)as described in Example 7. The reactions were then mixed with a TBE-Ureasample buffer and loaded on a 15% polyacrylamide TBE-Urea gel (Novex®,Invitrogen Cat. No. EC6885). As a qualitative as well as quantitativestandard, 1 pmol, 5 pmol and 10 pmol of the oligodeoxynucleotide whichwas used for the reassembling reaction, was loaded on the same gel. Thisgel was stained with SYBR®-Gold (Molecular Probes Cat. No. S-11494). TheSYBR®-Gold stain showed that the reassembled Qβ capsids containednucleic acid co-migrating with the oligodeoxynucleotides which were usedin the reassembly reaction. Taken together, resistance to benzonasedigestion of the nucleic acid content of the Qβ VLPs which had beenreassembled in the presence of oligodeoxynucleotides and isolation ofthe oligodeoxynucleotide from purified particles by proteinase Kdigestion, demonstrate packaging of the oligodeoxynucleotide.

Example 22

TABLE 2 Coupling of peptides derived from MelanA melanoma antigen to QbThe following MelanA peptide moieties were chemically synthesized: SEQAbbreviation* Sequence** ID NO: ELAGIGILTV 35 GHGHSYTTAE ELAGIGILTV 36SYTTAEELAGIGILTV ILGVL 37 ELAGIGILTVILGVL 38 MelanA 16-35 c GHGHSYTTAEEAAGIGILTV 39 MelanA 16-35 A/L c GHGHSYTTAE ELAGIGILTV 40 MelanA 26-35cgg EAAGIGILTV 41 MelanA 26-3 5 A/L cgg ELAGIGILTV 42 MelanA 20-40 A/L cSYTTAEELAGIGILTV ILGVL 43 MelanA 26-40 A/L cgg ELAGIGILTVILGVL 44 MelanA26-35-C A/L ELAGIGILTV ggc 45 CSPKSL-MelanA 26- CSPKSLELAGIGILTV 110 35A/L MelanA 26-40-C A/L ELAGIGILTVTLGVLGGC 111 * A/L indicates alanin tolysine exchange compared to the original wildtype MelanA peptide **amino acids from the linker sequence are indicated in small letters

The Following Procedures were Used for Chemical Coupling of the MelanAPeptide Moieties to Qb VLPs:

For peptide MelanA 16-35, MelanA 16-35 A/L and MelanA 26-35-C A/L: Asolution of 2 ml of 3.06 mg/ml Qb VLPs in 20 mM Hepes, pH 7.2 wasreacted for 30 minutes with 18.4 μl of a solution of 50 mM SMPH(succinimidyl-6-(β-maleimidopropionoamido hexanoate, Pierce) in DMSO at25° C. on a rocking shaker. The reaction solution was subsequentlydialyzed twice for 2 hours against 2 L of 20 mM Hepes, pH 72 at 4° C. 2ml of the dialyzed reaction mixture was then reacted with 18.4 μl of 50mM peptide stock solution (in DMSO) for two hours at 25° C. on a rockingshaker. The reaction mixture was subsequently dialyzed 2×2 hours against2 liters of 20 mM Hepes, pH 7.2 at 4° C. The coupled products were namedQb-MelanA 16-35 (SEQ ID NO: 39), Qb-MelanA 16-35 A/L (SEQ ID NO: 40) andQb-MelanA 26-35-C A/L (SEQ ID NO: 55). For MelanA 26-35: A solution of 2ml of 3.06 mg/ml Qb capsid protein in 20 mM Hepes, pH 7.2 was reactedfor 30 minutes with 75.3 μl of a solution of 50 mM SMPH in DMSO at 25°C. on a rocking shaker. The reaction solution was subsequently dialyzedtwice for 2 hours against 2 L of 20 mM Hepes, pH 7.2 at 4° C. 2 ml ofthe dialyzed reaction mixture was then reacted with 37.7 μl of 50 mMpeptide stock solution (in DMSO) for 4 hours at 25° C. on a rockingshaker. The reaction mixture was subsequently dialyzed 2×2 hours against2 liters of 20 mM Hepes, pH 7.2 at 4° C. The coupled product was namedQb-MelanA 26-35.

For MelanA 26-35 A/L (SEQ ID NO: 42): A solution of 2 ml of 3.06 mg/mlQb VLPs in 20 mM Hepes, pH 7.2 was reacted for 30 minutes with 37.7 μlof a solution of 50 mM SMPH in DMSO at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, pH 7.2 at 4° C. 2 ml of the dialyzed reaction mixturewas then reacted with 18.4 μl of 50 mM peptide stock solution (in DMSO)for 4 hours at 25° C. on a rocking shaker. The reaction mixture wassubsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, pH 7.2at 4° C. The coupled product was named Qb-MelanA 26-35 A/L.

For MelanA 20-40 A/L (SEQ ID NO: 43): A solution of 2 ml of 3.06 mg/mlQb VLPs in 20 mM Hepes, pH 7.2 was reacted for 30 minutes with 18.4 μlof a solution of 50 mM SMPH in DMSO at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, pH 7.2 at 4° C. 2 ml of the dialyzed reaction mixturewas then reacted with 184 μl of 5 mM peptide stock solution (in DMSO)for 4 hours at 25° C. on a rocking shaker. The reaction mixture wassubsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, pH 7.2at 4° C. The coupled product was named Qb-MelanA 20-40 A/L.

For MelanA 26-40 A/L (SEQ ID NO: 44): A solution of 2 ml of 3.06 mg/mlQb VLPs in 20 mM Hepes, pH 7.2 was reacted for 30 minutes with 37.7 μlof a solution of 50 mM SMPH in DMSO at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, pH 7.2 at 4° C. 2 ml of the dialyzed reaction mixturewas then reacted with 184 μl of 5 mM peptide stock solution (in DMSO)for 4 hours at 25° C. on a rocking shaker. The reaction mixture wassubsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, pH 7.2at 4° C. The coupled product was named Qb-MelanA 26-40 A/L.

Coupling efficiency was checked by SDS-PAGE analysis. FIG. 1 shows theSDS-PAGE analysis of Qb-MelanA VLPs. MelanA-peptides were coupled to QbVLPs. The final products were mixed with sample buffer and separatedunder reduced conditions on 16% Novex®Tris-Glycine gels for 1.5 hours at125 V. The separated proteins were stained by soaking the gel inCoomassie blue solution. Background staining was removed by washing thegel in 50% methanol, 8% acetic acid. The Molecular weight marker (P77085, New England BioLabs, Beverly, USA) was used as reference forQb-MelanA migration velocity (lane 1). 14 μg of either Qb alone (lane 2)or Qb derivatized with SMPH (lane 3) were loaded for comparison with 8μg of each final product: Qb-MelanA 16-35 (lane 4), Qb-MelanA 16-35 A/L(lane 5), Qb-MelanA 26-35 (lane 6) and Qb-MelanA 26-35 A/L (lane7).

The MelanA 16-35 A/L peptide contains the cytotoxic T lymphocyte (CTL)epitope MelanA 26-35 and Qb-MelanA 16-35 A/L was further studied for itsimmunogenicity in vitro and in vivo.

Example 23

Capacity of Immunostimulatory Sequences (ISS) to Activate Human Cells invitro

In order to select for the optimal ISS to be loaded in Qb-MelanAvaccine, series of CpG with different number of flanking Gs were testedfor their ability to upregulate CD69 on human CD8 T cells and to inducesecretion of IFN alpha and IL-12 in human PBMC.

Human PBMC were isolated from buffy coats and treated with the indicatedISS in RPMI medium containing 10% FCS for 18 h. IFN alpha in thesupernatants was measured by ELISA, using an antibody set provided byPBL Biomedical Laboratories. PBMC were stained with mouse anti-humanCD8-FITC, mouse anti-human CD19-PE and anti-human CD69-APC and analyzedby flow cytometry. G9-9 and G8-8 induced high levels of IFN alphasecretion (FIG. 2A). Decreasing the number of flanking Gs in the otheroligonucleotides resulted in lower IFN alpha secretion.

Treatment of PBMC with G9-9 and G8-8 upregulated CD69 on the cellmembrane of CD8 T cells to a nearly similar extend. Decreasing thenumber of flanking Gs (below 7) in the other oligonucleotides reducedtheir activity to induce secretion of IFN alpha (FIG. 2A) and toupregulate CD69 on T cells (FIG. 2B). These data show that G9-9 and G8-8have comparable high activity on human cells, therefore they can be usedas ISS in Qb-MelanA vaccine.

Example 24

Qbx33 VLPs Loaded with 03-6, or G6 Induces Protection Againstp33-Recombinant Vaccinia Virus Challenge

B6 mice were subcutaneously immunized with Qbx33 alone or loaded withG3-6 or G6 (see Examples 14 and 16). Eight days later, mice werechallenged with 1.5×106 pfu of recombinant Vaccinia virus, expressingthe LCMV-p33 antigen. After 4 days, mice were sacrificed and the viraltiters in ovaries were measured as previously described (Bachmann et al,Eur. J. Imunol. 1994, 24:2228). As depicted in FIG. 3, all micereceiving the Qbx33 vaccine loaded with either G3-6 or G6 were protectedfrom viral challenge. In contrast, naïve mice and mice immunized withQbx33 alone did not eliminate the virus from the ovaries. These datademonstrate that VLP alone is not sufficient to induce protective CTLimmune response, whereas VLP loaded with CpG are very efficient inpriming naïve CTL.

Similarly, immunization of mice with Qbx33 loaded with G8-8 is primingp33-specific CTL, as well as is inducing protection from recombinantVaccinia virus challenge.

Example 25

Q{tilde over (β)}MelanA 16-35 A/L VLPs are Processed and Presented bythe Human MHC Class I Allele HLA-A0201 and Induces Expansion ofFunctional MelanA-Specific CD8+T Cells in HLA-A2 Transgenic Mice

HHD mice express a chimeric monochain class I molecule with a humanβ2-microglobulin covalently linked to the N-terminus of A2 α1 and α2domains fused with Db α3 domain (Firat, H. et al 1999, Eur. J. Immunol.,29:3112). The HLA-A2 transgene expression in these mice allowsinvestigating the capacity of Q{tilde over (β)}MelanA 16-35 A/L VLPs tobe processed and presented as the CTL epitope MelanA 26-35 and to primeCTL in vivo. Furthermore, the effect of adjuvants, as ISS can be studiedin vivo.

HHD mice were either left untreated or immunized by injectingsubcutaneously with 100 μg Qb-MelanA 16-35 A/L or with Qb-pIC-MelanA16-35 A/L. Eight days later spleenocytes were isolated, resuspended inFACS buffer (PBS, 2% FCS, 5 mM EDTA, pH 8.2) and stained withHLA-A2-MelanA-PE labelled tetramers for 30 min at room temperature. In asecond step, rat anti-mouse CD8-APC (BD PharMingen, San Jose, USA) andanti mouse Mel14-FITC (BD PharMingen, San Jose, USA) were added for 30min at 4° C. After washing, erythrocytes were lysed with BD-LyzingSolution (BD Biosciences, San Jose, USA) for 10 min at room temperature.Finally, the spleen cells were analysed on a FACS Calibur usingCellQuest software. First of all, the cells were acquired in the forwardscatter and side scatter and the lymphocytes were gated. From thislymphocyte population, only the CD8 positive T cells were selected forfurther analyses. The HLA-A2-MelanA-PE and Mel14-FITC labelled cellswere measured with the FL2 and FL1 detector, respectively. The amount ofMelanA-specific, activated CD8+ T cells was calculated as percentHLA-A2-MelanA positive, Mel14 negative cells on total CD8+ lymphocytes.

Flow cytometry analysis showed that Qb-pIC-MelanA 16-35 A/L induced asurprisingly high expansion of MelanA-specific activated CD8+Mel14− Tcells (2.43% and 0.73%), which was higher compared to untreated animals(0.22% and 0.37%). It should be noted that the capacity of the vaccineincreased significantly only when Qb-MelanA was loaded with poly (I:C).

The human HLA-A2-MelanA tetramer does not bind very efficiently to mouseMelanA-specific T cells, as the protein is chimeric. Therefore we couldassume a much higher degree of antigen specific T cells in these mice.

In a similar experimental setting, immunization of HHD mice withQb-MelanA 16-35 A/L or Qb-MelanA 26-35 A/L loaded with G8-8 inducesexpansion of HLA-A2-MelanA-positive and Mel14 negative CD8 T cells.

Taken together these findings demonstrate the ability of ISS loadedQb-peptide vaccines to very efficiently prime CTL against foreign andself antigens.

Example 26

Coupling of gag-G50, nef-N56 and gag-G68n Peptide Antigen to Qβ VLP

The peptide gag-G50 (sequence: CQGQMVHQAISPRTLNAWVKAFSPEVIPMFSALSEGATPQDLNTMLNTVK) (SEQ ID NO: 86) and nef-N56 (sequence:CGVGFPVRPQVPLRPMTYKAAVDLSHFLKEKGGLE GPGIRYPLTFGWCFKLVPVEP) (SEQ ID NO:87) and gag-G68n (sequence: CGEIYKRWIILGLNKIVRMYQGQMVHQAISPRTLNAWVKAFSPEVIPMFSALSEGATPQDLNTMLNTVK) (SEQ ID NO: 88) were chemicallysynthesized. The peptides were ordered from the company SynPep, P.O. Box2999, Dublin, Calif. 94568, USA. Qβ VLP (Seq-ID No. 10) was then reactedat a concentration of 1.2 mg/ml (determined in a Bradford assay), with0.85 mM SMPH (Pierce) for 30 minutes at room temperature (RT). Thereaction mixture was then diafiltrated against 20 mM phosphate buffer pH7.2 and 50 mM MES pH 6.0 was added for gag-G50 coupling reactions, and50 mM Tris pH 8.5 for nef-N56 coupling reactions. A 5 mM stock ofpeptide was dissolved in DMSO and an equimolar amount TCEP was added tothe peptide in order to have reducing reaction conditions. Then, thederivatised Qβ particles reacted at a concentration of 1 mg/ml with0.214 mM gag-G50, 0.214 mM nef-N56 or 0.535 mM gag-G68n. Both peptides,gag-G50 and nef-N56, were also coupled under the same conditions, butfor the buffer, which was 50 mM Tris pH 8.5. The coupling reaction wasleft to proceed for 2 hours at 25° C.; samples were taken for SDS-PAGEanalysis, and the reaction mixtures dialyzed 2×2 hours against a1000-fold volume 20 mM phosphate, 0.05% Tween, pH 7.2. The dialyzedsamples were flash frozen in liquid nitrogen in aliquots for storage at−80° C. until further use. An aliquot was thawed, and coupling of theantigen to a Qβ subunit assessed by SDS-PAGE. The results of thecoupling reactions analyzed before the dialysis are shown in FIG. 4 andFIG. 5. Analysis of the dialyzed coupling reaction showed a similarpicture.

Coupling bands corresponding to one gag-G50 or nef-N56 peptide coupledper Qβ monomer or dimer are clearly visible demonstrating coupling ofboth peptides to the Qβ VLP.

Example 27

Coupling of HIV Peptides to Packaged Qβ VLP

Qβ VLP packaged with G8-8 oligonucleotide made as described in Example14 is coupled to HIV peptides as described in Example 26. The sequencesof the coupled peptides are gag-G50 (sequence:CQGQMVHQAISPRTLNAWVKAFSPEVIPMFSALSE GATPQDLNTMLNTVK) (SEQ ID NO: 86) andnef-N56 (sequence:CGVGFPVRPQVPLRPMTYKAAVDLSHFLKEKGGLEGPGIRYPLTFGWCFKLVPV EP) (SEQ ID NO:87) and gag-G68n (sequence:CGEIYKRWIILGLNKIVRMYQGQMVHQAISPRTLNAWVKAFSPEVIPMFSALSEG ATPQDLNTMLNTVK)(SEQ ID NO: 88). The resulting packaged and coupled Qβ VLP are analysedas described in Example 7 and in Example 14.

Example 28

Packaging of Qβ VLP Coupled to HIV Peptides

Qβ VLP is coupled to HIV peptides gag-G50, gag-G68n, or nef-N56 asdescribed in Example 26. Qβ VLP coupled either to gag-G50, gag-G68n, ornef-N56 is packaged with G8-8 oligonucleotide and analysed as describedin Example 7.

Example 29

Qbx33 Loaded with CpG can be Used in Homologous as Well in HeterologousPrime-Boost Regimen for the Induction of a Long Lasting Memory CD8+ TCell Response

Mice were immunized with 150 ug Qbx33/NKCpG and 8 days later thefrequencies of p33-specific T cells increased from 0.4%±0.2% in naïvemice to 7.5%±2.2% in immunized animals as measured with antigen.specificMHC/peptide tetramers. 20 days later the peptide specific CD8+ Tpopulation dropped down to 1.6%±0.7%. A second-imunisaiton of these mice30 days after the first inununisation with 150 ug Qbx33/NKPS could boostthe memory T cell response to up to 8.4%±1.9% specific T cells. Thisresponse dropped slowly down but could be boosted again 4 months afterthe first boost with 150 ug Qbx33/NKPS reaching T cell levels of23.8%±5.2%.

When 3 mice were primed with 50 ug p33 peptide mixed with 20 nmol NKPSand IFA only 0.6%±_(—)0.4% specific CD8+ T cells could be induced untilday 8 post-immunisation. Nevertheless, this low response could beboosted efficiently 7 weeks later with Qbx33/NKPS to levels of28.5%±9.8%. Immunisation with 1×10 exp 6 plaque forming units ofrecombinant vaccinia virus expressing the p33-peptide could hardlyinduce any T cell response (1.1%±0.5%) but was boosted very efficientlyboosted 6 months later with 150 ug Qbx33/NKPS to T cells levels of28.1±_(—)4.2%.

These results show, that Qb loaded with CpG very efficiently boosts anypre-existing T cell response in heterologous as well as homologous primeboost regimens. It should be noted, that Qb/NKPS can even boost a veryinefficiently primed T cell response with peptides or recombinantviruses. In addition, when a strong T cell response was established withQbx33/NKPS we were able to boost this response using an immunologicallyeffective amount of a heterologous vaccine such as the p33 peptidealone, recombinant virus expressing p33, or p33 fused or coupled to aVLP. In the latter, the used VLP is not a VLP derived from RNA phage Qbbut e.g. HBcAg or VLP derived from AP205.

1-68. (canceled)
 69. A method of producing a composition for enhancingan immune response in an animal, said composition comprising avirus-like particle and an immunostimulatory substance packaged intosaid virus-like particle, wherein said method comprises: (a)disassembling said virus-like particle; (b) adding saidimmunostimulatory substance; and (c) reassembling said virus-likeparticle; wherein said immunostimulatory substance is an unmethylatedCpG-containing oligonucleotide, wherein the CpG motif of saidunmethylated CpG-containing oligonucleotide is part of a palindromicsequence, and wherein said palindromic sequence is flanked at its3′-terminus and at its 5′-terminus by less than 10 guanosine entities.70. The method of claim 69, wherein said unmethylated CpG-containingoligonucleotide consists of 10 to 30 nucleotides, and wherein saidpalindromic sequence is GACGATCGTC (SEQ ID NO:1), and wherein saidpalindromic sequence is flanked at its 5′-terminus by at least 3 and atmost 9 guanosine entities and wherein said palindromic sequence isflanked at its 3′-terminus by at least 6 and at most 9 guanosineentities. 71-72. (canceled)
 73. The method of claim 70, wherein saidunmethylated CpG-containing oligonucleotide consists of a nucleic acidsequence selected from the group consisting of: (a) GGGGACGATCGTCGGGGGG;(SEQ ID NO: 2) (b) GGGGGACGATCGTCGGGGGG; (SEQ ID NO: 3) (c)GGGGGGACGATCGTCGGGGGG; (SEQ ID NO: 4) (d) GGGGGGGACGATCGTCGGGGGG; (SEQID NO: 5) (e) GGGGGGGGACGATCGTCGGGGGGG; (SEQ ID NO: 6) (f)GGGGGGGGGACGATCGTCGGGGGGGG; (SEQ ID NO: 7) (g)GGGGGGGGGGACGATCGTCGGGGGGGGG; (SEQ ID NO: 8) and (h)GGGGGGCGACGACGATCGTCGTCGGGGGGG. (SEQ ID NO: 9)


74. The method of claim 69, wherein said unmethylated CpG-containingoligonucleotide consists of 10 to 30 nucleotides, wherein saidpalindromic sequence is GACGATCGTC (SEQ ID NO:1), and wherein saidpalindromic sequence is flanked at its 5′-terminus by at least 4 and atmost 9 guanosine entities and wherein said palindromic sequence isflanked at its 3′-terminus by at least 6 and at most 9 guanosineentities. 75-79. (canceled)
 80. The method of claim 69 furthercomprising removing nucleic acids of said disassembled virus-likeparticle.
 81. The method of claim 69 further comprising purifying saidcomposition after reassembly.
 82. The method of claim 69, furthercomprising binding an antigen or antigenic determinant to saidvirus-like particle.
 83. (canceled)
 84. The method of claim 82, whereinsaid antigen or antigenic determinant is bound to said virus-likeparticle after reassembling said virus-like particle. 85-113. (canceled)114. The method of claim 69, wherein said virus-like particle is avirus-like particle of an RNA-phage.
 115. The method of claim 114,wherein said RNA-phage is bacteriophage Qβ.
 116. The method of claim115, wherein said virus-like particle comprises or consists ofrecombinant proteins of RNA-bacteriophage Qβ, wherein said recombinantproteins consist of the amino acid sequence of SEQ ID NO:10.
 117. Themethod of claim 116, wherein said unmethylated CpG-containingoligonucleotide consists of a nucleic acid sequence selected from thegroup consisting of: (a) GGGGGGACGATCGTCGGGGGG; (SEQ ID NO: 4) (b)GGGGGGGACGATCGTCGGGGGG; (SEQ ID NO: 5) (c) GGGGGGGGACGATCGTCGGGGGGG;(SEQ ID NO: 6) and (d) GGGGGGGGGACGATCGTCGGGGGGGG. (SEQ ID NO: 7)


118. The method of claim 115, further comprising binding an antigen orantigenic determinant to said virus-like particle by way of a covalentbond.
 119. The method of claim 118, wherein said virus-like particlecomprises at least one first attachment site and wherein said antigen orantigenic determinant further comprises at least one second attachmentsite selected from the group consisting of: (a) an attachment site notnaturally occurring with said antigen or antigenic determinant; and (b)an attachment site naturally occurring with said antigen or antigenicdeterminant; wherein said binding of said antigen or antigenicdeterminant to said virus-like particle is effected through associationbetween said first attachment site and said second attachment site,wherein said association is through at least one non-peptide covalentbond, and wherein said antigen or antigenic determinant and saidvirus-like particle interact through said association to form an orderedand repetitive antigen array.
 120. The method of claim 119, wherein saidfirst attachment site is a lysine residue and said second attachmentsite is a cysteine residue.
 121. The method of claim 120, wherein saidbinding is effected by a hetero-bifunctional crosslinker.
 122. Themethod of claim 121, wherein said virus-like particle comprises orconsists of recombinant proteins of RNA-bacteriophage Qβ, wherein saidrecombinant proteins consist of the amino acid sequence of SEQ ID NO:10.123. The method of claim 122, wherein said unmethylated CpG-containingoligonucleotide consists of 10 to 30 nucleotides, wherein saidpalindromic sequence is GACGATCGTC (SEQ ID NO:1), wherein saidpalindromic sequence is flanked at its 5′-terminus by at least 3 and atmost 9 guanosine entities and wherein said palindromic sequence isflanked at its 3′-terminus by at least 6 and at most 9 guanosineentities.
 124. The method of claim 123, wherein said unmethylatedCpG-containing oligonucleotide consists of a nucleic acid sequenceselected from the group consisting of (a) GGGGGGACGATCGTCGGGGGG; (SEQ IDNO: 4) (b) GGGGGGGACGATCGTCGGGGGG; (SEQ ID NO: 5) (c)GGGGGGGGACGATCGTCGGGGGGG; (SEQ ID NO: 6) and (d)GGGGGGGGGACGATCGTCGGGGGGGG. (SEQ ID NO: 7)


125. The method of claim 124, wherein said hetero-bifunctionalcross-linker is succinimidyl-6-(β-maleimidopropionamido)hexanoate(SMPH).